Compounds and pharmaceutical compositions for the treatment of liver disorders

ABSTRACT

Provided herein are compounds, compositions and methods for the treatment of liver disorders, including liver cancer and metabolic diseases, such as diabetes, hyperlipidemia, atherosclerosis, and obesity. Specifically, compounds and compositions of nucleoside derivatives are disclosed, which can be administered either alone or in combination with other anti-cancer agents.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This patent applications claims the benefit of priority under 35 U.S.C.§ 119 to 1) U.S. Provisional Appl. No. 60/877,944, filed Dec. 28, 2006;2) U.S. Provisional Appl. No. 60/936,290, filed Jun. 18, 2007; and 3)U.S. Provisional Application No. 60/985,891, filed Nov. 6, 2007. Thedisclosures of the above referenced applications are incorporated byreference in their entirety herein.

2. FIELD

The present invention relates to compounds, methods and pharmaceuticalcompositions, for use in treatment and prevention of disorders of theliver, including cancer.

3. BACKGROUND

Drug induced toxicities and pharmacological side effects are oftenassociated with interactions by the drug or drug metabolite in tissuesnot associated with the pharmacological benefits of the drug therapy. Inother cases, the desired pharmacological effect is poorly achievedeither because of dose-limiting toxicities or inadequate drug levels inthe target tissues. Thus, there is a need to deliver drugs to specifictissues or organs. High organ specificity can be achieved by a varietyof mechanisms including local administration to the target organ anddrug-protein conjugates. Local administration to the target organ is aninvasive procedure. Drug-protein conjugates exhibit poor oralbioavailability, limitations in carrier manufacturing and drug loading,a potential for diminished liver uptake due to down regulation of thereceptor in diseased tissue, and a high incidence of antibody induction.A third approach entails use of prodrugs that are activated by enzymeshighly enriched in the target organ.

There is a particular need to deliver drugs to the liver to treatdiseases such as cancer and metabolic disorders. Many therapies forthese conditions have narrow therapeutic indices and many therapeuticindications could be benefitted by selective delivery of the therapeuticagent to the liver.

4. SUMMARY

Phosphoroamidate and phosphonoamidate compound forms of a variety oftherapeutic agents are provided, as well as methods for theirmanufacture and use in the treatment of a variety of disorders includingliver cancer, inflammation, fibrosis and metabolic disorders. In oneembodiment, the compound is a S-pivaloyl-2-thioethyl phosphoroamidate,S-pivaloyl-2-thioethyl phosphonoamidate, S-hydroxypivaloyl-2-thioethylphosphoroamidate or S-hydroxypivaloyl-2-thioethyl phosphonoamidate. Asused herein, a “phosphoroamidate or phosphonoamidate compound of atherapeutic agent” includes a therapeutic agent derivatized to include aphosphoroamidate or phosphonoamidate group. The therapeutic agent is,for example, an anti-cancer agent that includes, or has been derivatizedto include, a reactive group, such as a hydroxyl, for attachment of thephosphoroamidate or phosphonoamidate moiety. Such therapeutic agentsinclude, but are not limited to nucleosides and nucleoside analogsincluding acyclic nucleosides. In some embodiments, phosphoroamidate orphosphonoamidate compounds of nucleotides and nucleotide analogs, suchas 2′-branched and 4′-branched nucleosides are provided. Such compoundscan be administered in an effective amount for the treatment of liverdisorders, including cancer.

In certain embodiments, while not being limited to any theory, it ispossible that the parent drug is obtained from selective metabolism ofthe phosphoroamidate or phosphonoamidate compound in the liver and thusthe parent drug provided herein is capable of accumulating in the liver.Accordingly, provided are methods of directing phosphoroamidate orphosphonoamidate compounds disclosed herein to the liver.

In certain embodiments, phosphoroamidate or phosphonoamidate compoundsof pharmaceutical agents for the treatment of a liver disorder can bemade and used therapeutically as described herein. A variety ofphosphoroamidate or phosphonoamidate compounds can be used in thetreatment of liver disorders. In particular, therapeutic agents for thetreatment of liver cancer can be derivatized to form a phosphoroamidateor phosphonoamidate compound as described herein, and used for thetreatment of liver cancers. Liver cancers that can be treated includebenign tumors, malignant tumors, hemangioma, hepatic adenomas, focalnodular hyperplasia, hepatocellular carcinoma, fibrolamellar carcinoma,cholangiocarcinomas, bile duct cancers, and other primary and metastaticcancers of the liver.

Phosphoroamidate and phosphonoamidate compounds of a variety oftherapeutic agents are provided. The compounds can be formed usingmethods available in the art and those disclosed herein. Such compoundscan be used in some embodiments to enhance delivery of the drug to theliver. In one embodiment, the compound comprises an S-acyl-2-thioethylphosphoroamidate or an S-acyl-2-thioethyl phosphonoamidate derivative,e.g., a S-pivaloyl-2-thioethyl phosphoroamidate or aS-hydroxypivaloyl-2-thioethyl phosphonoamidate derivative.

In some embodiments, the phosphoroamidate or phosphonoamidate compounds,as well as salts thereof, and compositions comprising the compounds,provided herein are useful for treatment of disorders of the liver,including cancer. In other embodiments, the phosphoroamidate orphosphonoamidate compounds, as well as salts thereof, and compositionscomprising the compounds, provided herein are useful for treatment ofmetabolic diseases, such as diabetes, hyperlipidemia, atherosclerosis,and obesity. In other embodiments, the compounds, as well as saltsthereof, and compositions comprising the compounds, provided herein areuseful for treatment of liver fibrosis and inflammation.

In one embodiment, the compound provided herein is a compound of FormulaI:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof, wherein

X^(a) is

Z is O or S;

each W is independently O or S;

R^(y) and R^(u) each independently represent alkyl, alkenyl, alkynyl,aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl, alkoxy,heterocyclyl, or heteroaryl, all optionally substituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring;

n is 0-3; n2 is 1-4; and

R¹ is a moiety derivable by removal of a hydrogen from a group, such asa hydroxy group, of a therapeutic agent such as an anti-cancer drug.

In another embodiment,

Z is O, S, NH or NR^(w), where R^(w) is, e.g., alkyl, alkyl, alkenyl,alkynyl, aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl,alkoxy, heterocyclyl, or heteroaryl, all optionally substituted;

each W is O, S, NH or NR^(w), where R^(w) is, e.g., alkyl, alkyl,alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino,aminoalkyl, alkoxy, heterocyclyl, or heteroaryl, all optionallysubstituted;

R^(y) and R^(u) each independently represent alkyl, alkenyl, alkynyl,aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl, alkoxy,heterocyclyl, or heteroaryl, all optionally substituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring;

n is 0-3; n₂ is 1-4; and

R¹ is a moiety derivable by removal of a hydrogen from a group, such asa hydroxy group, of a therapeutic agent such as an anti-cancer drug.

Those of skill in the art will recognize that compounds of Formula I canbe designed or prepared by reaction, e.g., at a hydroxy group of saiddrug, for example via condensation or dehydration. For convenience, inthe description herein when exemplary substituents, such as R¹ groupsare identified as a drug in a phosphoroamidate or phosphonoamidatecompound, e.g. in a formula, those of skill in the art will recognizethat the compound comprises a derivative, e.g. a radical of theanti-cancer drug. Those derivatives can for example be prepared byelimination of a hydrogen radical from a hydroxy group of the drug, forinstance in a dehydration reaction.

In certain embodiments of Formula I, R¹ is a nucleoside comprising acyclic or acyclic sugar or an analog thereof.

In certain embodiments, R¹ is an anti-cancer drug selected fromclarubicin, decitabine, daunorubicin, dihydro-5-azacytidine,doxorubicin, epirubicin, estramustin, etoposide, fludarabine,7-hydroxychlorpromazin, neplanocin A, podophyllotoxin, tezacitabine,troxacitabine, vinblastin, vincristin, vindesin, etoposide, teniposide,NK-611, camptothecin, irinotecan, 9-aminocamptothecin, GG-211,topotecan, paclitaxel, Azatoxin, coformycin, pirarubicin, nelarabine andlosoxantrone.

In one embodiment, R¹ is an immunosuppressant, such as pentostatin,combretastatin A-4, mycophenolic acid or mitoxantrone.

In certain embodiments according to formula I, R^(y) is substitutedalkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b) areindependently hydrogen, alkyl, substituted alkyl, benzyl or substitutedbenzyl, for instance hydroxy- or amino-substituted alkyl or benzyl.

In another embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) whereeach R^(c) is independently alkyl, substituted alkyl, aryl orsubstituted aryl, for instance hydroxy- or amino-substituted alkyl oraryl; and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl.

In a further embodiment, R^(a) and R^(b) are independently benzyl orsubstituted alkyl. In a further embodiment, R^(y) is selected from thegroup consisting of alkyl and hydroxyalkyl. In certain embodiments,R^(y) is —C(CH₃)₂CH₂OH.

In certain embodiments, the compounds provided herein are selected suchthat R¹ is not 3′-azido-2′,3′-dideoxythymidine.

In another embodiment, the compound provided herein is a compound ofFormula IIa or IIb:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof, wherein

R^(y) is alkyl, alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl,cycloalkenyl, amino, aminoalkyl, heterocyclyl or heteroaryl, alloptionally substituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring; and

R¹ is a drug such as an anti-cancer drug.

In certain embodiments according to Formula IIa or IIb, R^(y) issubstituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b)are each independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In another embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c)where each R^(c) is independently alkyl, substituted alkyl, aryl orsubstituted aryl, for instance hydroxy- or amino-substituted alkyl oraryl; and R^(a) and R^(b) are independently hydrogen, alkyl, substitutedalkyl, benzyl or substituted benzyl, for instance hydroxy- oramino-substituted alkyl or benzyl. In a further embodiment, R^(a) andR^(b) are each independently benzyl or substituted alkyl. In a furtherembodiment, R^(y) is selected from the group consisting of alkyl andhydroxyalkyl. In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In some embodiments, provided herein are:

-   (a) compounds as described herein, e.g. of Formula I, IIa or IIb,    and pharmaceutically acceptable salts and compositions thereof;-   (b) compounds as described herein, e.g. of Formula I, IIa or IIb,    and pharmaceutically acceptable salts and compositions thereof for    use in the treatment and/or prophylaxis of a liver disorder;-   (c) processes for the preparation of compounds as described herein,    e.g. of Formula I, IIa or IIb, as described in more detail below;-   (d) pharmaceutical formulations comprising a compound as described    herein, e.g. of Formula I, IIa or IIb, or a pharmaceutically    acceptable salt thereof together with a pharmaceutically acceptable    carrier or diluent; and-   (e) pharmaceutical formulations comprising a compound as described    herein, e.g. of Formula I, IIa or IIb, or a pharmaceutically    acceptable salt thereof together with one or more other effective    anti-cancer agents, optionally in a pharmaceutically acceptable    carrier or diluent.

In certain embodiments, the following phosphoroamidate andphosphonoamidate formulas and compounds are provided, which optionallyact as thyroid hormone receptor effectors:

wherein

each R, if present, is independently alkyl, halogen or hydroxyl;

X, if present, is CH₂, O or S;

R^(y), if present, is optionally substituted alkyl, wherein thesubstituted alkyl is optionally hydroxyalkyl or aminoalkyl, e.g.,—C(CH₃)₂CH₂OH; and

R^(a) and R^(b), if present, are independently hydrogen; unsubstitutedalkyl; or alkyl substituted with aryl, amino, amido, hydroxyl, alkoxy,aminoalkyl, hydroxyalkyl, aryl, or heteroaryl, each optionallysubstituted; wherein, in one embodiment, R^(a) and R^(b) areindependently H or a benzyl that is optionally substituted, for example,with hydroxy or amino.

In certain embodiments according to formula IIIa or b, IVa or b, Va orb, VIa or b, VIIa or b, VIII a or b, R^(a) is hydrogen, R^(b) is—CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In certain embodiments, the thyroid hormone receptor effector compoundprovided herein has a formula selected from:

wherein

R^(x) and R^(z) are each independently hydrogen or alkyl;

R^(w) is alkyl;

R^(y) is alkyl, alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl,cycloalkenyl, amino, aminoalkyl, heterocyclyl or heteroaryl, alloptionally substituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring.

In certain embodiments, the thyroid hormone receptor effector providedherein is selected from:

In certain embodiments, R^(a) is hydrogen, R^(b) is —CH₂—C₆H₅ and R^(y)is —C(CH₃)₂CH₂OH.

In certain embodiments, the compound or Formula selected from IIIa or b,IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b is derived from aphosphonate compound useful for inhibiting gluconeogenesis, optionallyby inhibiting the enzyme fructose 1,6-bisphosphatase (FBPase).

In certain embodiments, the compound or Formula selected from IXa or b,Xa or b, XIa or b, XIIa or b, XIIIa or b and XIVa or b is derived from acompound useful for inhibiting gluconeogenesis, optionally by inhibitingthe enzyme fructose 1,6-bisphosphatase (FBPase).

In certain embodiments, the compound or Formula selected from IIIa or b,IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b is a phosphonicacid-containing compound that binds to a thyroid receptor in the liver,and is optionally an agonist, antagonist, partial agonist or partialantagonist of T3. Inhibition of gluconeogenesis can result in bloodglucose lowering in diabetic subjects. Such compounds can exhibitenhanced pharmacokinetics including oral bioavailability and liver druglevels.

In certain embodiments, provided is a method of treatment of a subjectin need thereof, the method comprising administering to the subject aphosphoroamidate and phosphonoamidate compound or Formula selected fromIIIa or b, IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b, IXa orb, X a or b, XIa or b, XIIa or b, XIIIa or b and XIVa or b or apharmaceutically acceptable salt, enantiomer, ester or prodrug thereof,in an amount effective for one or more of the following:

reducing plasma lipid levels, lowering cholesterol levels, reducingtriglyceride levels, or increasing the ratio of HDL to LDL;

lowering blood glucose levels;

treating hyperlipidemia or hypercholesterolemia;

treating obesity, reducing fat content, treating fatty liver, reducingweight or preventing weight gain;

treating atherosclerosis, coronary heart disease, heart failure,nephrotic syndrome, or chronic renal failure;

lowering blood glucose levels, treating diabetes, impaired glucosetolerance, metabolic syndrome x, insulin resistance or hyperinsulinemia;

increasing levels of genes associated with gluconeogenesis;

decreasing hepatic glycogen levels or maintaining or improving glycemiccontrol;

amelioration of hyperinsulinemia and/or decrease of glucose levels indiabetic subjects at doses that optionally do not affect cardiacfunction, e.g., heart rate, force of systolic contraction, duration ofdiastolic relaxation, vascular tone, or heart weight;

treating thyroid disease, thyroid cancer, depression, glaucoma, cardiacarrhythmias, heart failure, or osteoporosis;

increasing mitochondrial biogenesis, or increasing expression of PGC-1,AMP activated protein kinase or nuclear respiratory factor;

inhibiting hepatic gluconeogenesis; or

modulating expression of certain genes in the liver resulting in effectson lipids (e.g., cholesterol), glucose, lipoproteins, and triglycerides,or modulation of T3-responsive genes.

In certain embodiments, the compounds do not affect thyroid function,thyroid production of circulating iodinated thyronines such as T3 andT4, and/or the ratio of T3 to T4.

Also provided are pharmaceutical compositions comprising the compounds,e.g., in a dosage unit suitable for administration, e.g., oraladministration.

5. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts depletion of B184 (NM108 hydroxySATE phosphoroamidate)after incubation with and without NADPH in monkey liver S9.

FIG. 2 depicts depletion of B102 (NM107 hydroxySATE phosphoroamidate)after incubation with and without NADPH in monkey liver S9.

6. DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein are compounds, compositions and methods useful fortreating liver disorders, such as cancer, or metabolic diseases, such asdiabetes, hyperlipidemia, atherosclerosis, and obesity. Further providedare dosage forms useful for such methods.

6.1 Definitions

When referring to the compounds provided herein, the following termshave the following meanings unless indicated otherwise.

The term “alkyl”, as used herein, unless otherwise specified, includes asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of typically C₁ to C₁₀, and specifically includes methyl,CF₃, CCl₃, CFCl₂, CF₂Cl, ethyl, CH₂CF₃, CF₂CF₃, propyl, isopropyl,cyclopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl, cyclopentyl,isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The termincludes both substituted and unsubstituted alkyl groups, andparticularly includes halogenated alkyl groups, and even moreparticularly fluorinated alkyl groups. Non-limiting examples of moietieswith which the alkyl group can be substituted are selected from thegroup consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et al., Protective Groups inOrganic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term “lower alkyl”, as used herein, and unless otherwise specified,includes a C to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted moieties.

“Alkylene” includes divalent saturated aliphatic hydrocarbon groupsparticularly having up to about 11 carbon atoms and more particularly 1to 6 carbon atoms which can be straight-chained or branched. This termis exemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkenyl” includes monovalent olefinically unsaturated hydrocarbongroups, in certain embodiment, having up to about 11 carbon atoms, from2 to 8 carbon atoms, or from 2 to 6 carbon atoms, which can bestraight-chained or branched and having at least 1 or from 1 to 2 sitesof olefinic unsaturation. Exemplary alkenyl groups include ethenyl(—CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl (—C(CH₃)═CH₂), vinyl andsubstituted vinyl, and the like.

“Alkenylene” includes divalent olefinically unsaturated hydrocarbongroups, in certain embodiments, having up to about 11 carbon atoms orfrom 2 to 6 carbon atoms which can be straight-chained or branched andhaving at least 1 or from 1 to 2 sites of olefinic unsaturation. Thisterm is exemplified by groups such as ethenylene (—CH═CH—), thepropenylene isomers (e.g., —CH═CHCH₂— and —C(CH₃)═CH— and —CH═C(CH₃)—)and the like.

“Alkynyl” includes acetylenically unsaturated hydrocarbon groups, incertain embodiments, having up to about 11 carbon atoms or from 2 to 6carbon atoms which can be straight-chained or branched and having atleast 1 or from 1 to 2 sites of alkynyl unsaturation. Non-limitingexamples of alkynyl groups include acetylenic, ethynyl (—C≡CH),propargyl (—CH₂C≡CH), and the like.

The term “aryl”, as used herein, and unless otherwise specified,includes phenyl, biphenyl, or naphthyl, and preferably phenyl. The termincludes both substituted and unsubstituted moieties. The aryl group canbe substituted with any described moiety, including, but not limited to,one or more moieties selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), alkyl, hydroxyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991.

“Alkoxy” includes the group —OR where R is alkyl. Particular alkoxygroups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

“Alkoxycarbonyl” includes a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Amino” includes the radical —NH₂.

“Carboxyl” includes the radical —C(O)OH.

The term “alkylamino” or “arylamino” includes an amino group that hasone or two alkyl or aryl substituents, respectively. Unless otherwisespecifically stated in this application, when alkyl is a suitablemoiety, lower alkyl is preferred. Similarly, when alkyl or lower alkylis a suitable moiety, unsubstituted alkyl or lower alkyl is preferred.

“Halogen” or “halo” includes chloro, bromo, fluoro or iodo.

“Monoalkylamino” includes the group alkyl-NR′—, wherein R′ is selectedfrom hydrogen and alkyl.

“Thioalkoxy” includes the group —SR where R is alkyl.

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, nitrogen, or phosphorus atom toprevent its further reaction or for other purposes. A wide variety ofoxygen and nitrogen protecting groups are known to those skilled in theart of organic synthesis.

“Pharmaceutically acceptable salt” includes any salt of a compoundprovided herein which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Such salts may bederived from a variety of organic and inorganic counter-ions well knownin the art. Such salts include: (1) acid addition salts formed withorganic or inorganic acids such as hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic,propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic,lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric,tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric,cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2)salts formed when an acidic proton present in the parent compound either(a) is replaced by a metal ion, e.g., an alkali metal ion, an alkalineearth ion or an aluminum ion, or alkali metal or alkaline earth metalhydroxides, such as sodium, potassium, calcium, magnesium, aluminum,lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with anorganic base, such as aliphatic, alicyclic, or aromatic organic amines,such as ammonia, methylamine, dimethylamine, diethylamine, picoline,ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylene-diamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like.

Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium and the like, and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrohalides, e.g. hydrochloride andhydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate,trifluoroacetate, trichloroacetate, propionate, hexanoate,cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate,cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate,benzenesulfonate (besylate), 4-chlorobenzenesulfonate,2-naphthalenesulfonate, 4-toluenesulfonate, camphorate,camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate,glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate,lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,salicylate, stearate, cyclohexylsulfamate, quinate, muconate and thelike.

The term “alkaryl” or “alkylaryl” includes an aryl group with an alkylsubstituent. The term aralkyl or arylalkyl includes an alkyl group withan aryl substituent.

The term “purine” or “pyrimidine” base includes, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-alkylaminopurine, N⁶-thioalkyl purine, N²-alkylpurines,N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil,C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine,C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-iodopyrimidine, C⁶-iodo-pyrimidine, C⁵—Br-vinyl pyrimidine,C⁶—Br-vinyl pyrimidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Purine bases include, but are not limited to,guanine, adenine, hypoxanthine, 7-deazaguanine, 7-deazaadenine,2,6-diaminopurine, and 6-chloropurine. Functional oxygen and nitrogengroups on the base can be protected as necessary or desired. Suitableprotecting groups are well known to those skilled in the art, andinclude trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, andt-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such asacetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.

The term “acyl” or “O-linked ester” includes a group of the formulaC(O)R′, wherein R′ is an straight, branched, or cyclic alkyl (includinglower alkyl), carboxylate reside of amino acid, aryl including phenyl,alkaryl, arylalkyl including benzyl, alkoxyalkyl includingmethoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted alkyl(including lower alkyl), aryl including phenyl optionally substitutedwith chloro, bromo, fluoro, iodo, C₁ to C₄ alkyl or C₁ to C₄ alkoxy,sulfonate esters such as alkyl or arylalkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxy-trityl, substituted benzyl, alkaryl, arylalkyl includingbenzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such asphenoxymethyl. Aryl groups in the esters optimally comprise a phenylgroup. In particular, acyl groups include acetyl, trifluoroacetyl,methylacetyl, cyclpropylacetyl, propionyl, butyryl, hexanoyl, heptanoyl,octanoyl, neo-heptanoyl, phenylacetyl, 2-acetoxy-2-phenylacetyl,diphenylacetyl, α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl,chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl,tert-butylacetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl,7H-dodecafluoro-heptanoyl, perfluoro-heptanoyl,7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nonafluoro-3,6-dioxa-heptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxy-benzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,1-pyrrolidinecarbonyl, 4-phenylbenzoyl.

The term “amino acid” includes naturally occurring and synthetic α, β γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” with respect to a nucleoside composition includes anucleoside composition that includes at least 85 or 90% by weight,preferably 95%, 98%, 99% or 100% by weight, of the designated enantiomerof that nucleoside. In a preferred embodiment, in the methods andcompounds of this invention, the compounds are substantially free ofenantiomers.

Similarly, the term “isolated” with respect to a nucleoside compositionincludes a nucleoside composition that includes at least 85, 90%, 95%,98%, 99% to 100% by weight, of the nucleoside, the remainder comprisingother chemical species or enantiomers.

“Solvate” includes a compound provided herein or a salt thereof, thatfurther includes a stoichiometric or non-stoichiometric amount ofsolvent bound by non-covalent intermolecular forces. Where the solventis water, the solvate is a hydrate.

As used herein, the terms “subject” and “patient” are usedinterchangeably herein. The terms “subject” and “subjects” refer to ananimal, such as a mammal including a non-primate (e.g., a cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as acynomolgous monkey, a chimpanzee and a human), and for example, a human.In one embodiment, the subject is refractory or non-responsive tocurrent treatments for cancer. In another embodiment, the subject is afarm animal (e.g., a horse, a cow, a pig, etc.) or a pet (e.g., a dog ora cat). In one embodiment, the subject is a human.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment or preventionof a disorder or one or more symptoms thereof. In certain embodiments,the term “therapeutic agent” includes a compound provided herein. In oneembodiment, a therapeutic agent is an agent which is known to be usefulfor, or has been or is currently being used for the treatment orprevention of a disorder or one or more symptoms thereof.

“Therapeutically effective amount” includes an amount of a compound orcomposition that, when administered to a subject for treating a disease,is sufficient to effect such treatment for the disease. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the disease and its severity, and the age, weight, etc.,of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating a disease or disorder that exists in asubject. In another embodiment, “treating” or “treatment” includesameliorating at least one physical parameter, which may be indiscernibleby the subject. In yet another embodiment, “treating” or “treatment”includes modulating the disease or disorder, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In yet anotherembodiment, “treating” or “treatment” includes delaying the onset of thedisease or disorder.

As used herein, the terms “prophylactic agent” and “prophylactic agents”as used refer to any agent(s) which can be used in the prevention of adisorder or one or more symptoms thereof. In certain embodiments, theterm “prophylactic agent” includes a compound provided herein. Incertain other embodiments, the term “prophylactic agent” does not refera compound provided herein. For example, a prophylactic agent is anagent which is known to be useful for, or has been or is currently beingused to the prevent or impede the onset, development, progression and/orseverity of a disorder.

As used herein, the phrase “prophylactically effective amount” includesthe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention or reduction of the development, recurrenceor onset of one or more symptoms associated with a disorder (, or toenhance or improve the prophylactic effect(s) of another therapy (e.g.,another prophylactic agent).

6.2 Exemplary Embodiments

6.2.1 Compounds

Phosphoroamidate and phosphonoamidate compounds of a variety oftherapeutic agents can be formed using methods available in the art andthose disclosed herein. Such compounds can be used in some embodimentsto enhance delivery of the drug to the liver. In one embodiment, thecompound is an S-acyl-2-thioethyl phosphoroamidate or anS-acyl-2-thioethyl phosphonoamidate derivative, e.g., aS-pivaloyl-2-thioethyl phosphoroamidate or aS-hydroxypivaloyl-2-thioethyl phosphonoamidate. Therapeutic agents thatcan be derivatized to compound form include an anti-cancer agent thatincludes, or has been derivatized to include a reactive group forattachment of the phosphoroamidate or phosphonoamidate moiety, includingbut not limited to nucleosides and nucleoside analogues includingacyclic nucleosides.

Phosphoroamidate or phosphonoamidate compound forms of a variety ofnucleosides can be formed from nucleosides disclosed herein andavailable in the art. In particular, anti-cancer nucleosides can bederivatized to form a phosphoroamidate or phosphonoamidate compound thatcan enhance delivery to the liver.

In one embodiment, the phosphoroamidate or phosphonoamidate compoundprovided herein is a compound of formula IIa or IIb:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof, wherein;

R^(y) is alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,cycloalkenyl, amino, heterocyclyl or heteroaryl, all optionallysubstituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, arylalkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring; and

R¹ is a drug such as an anti-cancer drug.

In certain embodiments, the compound of formula IIa or IIb is selectedwith a proviso that when R^(y) is tert-butyl or hydroxy-tert-butyl, thenR¹ is not 3′-azido-2′,3′-dideoxythymidine.

In certain embodiments, R¹, R^(a), R^(b) and R^(y) are optionallysubstituted with one or more substituents as defined herein, e.g., inthe definitions.

In certain embodiments, the compounds are of Formula IIa or IIb, whereinR^(y) is alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,cycloalkenyl, amino, heterocyclyl or heteroaryl;

R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, arylalkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; and

R¹ is an anti-cancer drug.

In one embodiment, R¹ or R¹—CH₂— is a nucleoside comprising a cyclic oracyclic sugar or analog thereof, including any nucleoside or analoguethereof described herein or known in the art.

In certain embodiments of Formula IIa or IIb, R^(y) is substitutedalkyl, e.g. hydroxyalkyl or aminoalkyl; and R^(a) and R^(b) areindependently hydrogen, alkyl, substituted alkyl, benzyl or substitutedbenzyl, for instance hydroxy- or amino-substituted alkyl or benzyl. Inanother embodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where eachR^(c) is independently alkyl, substituted alkyl, aryl or substitutedaryl, for instance hydroxy- or amino-substituted alkyl or aryl; andR^(a) and R^(b) are independently hydrogen, alkyl, substituted alkyl,benzyl or substituted benzyl, for instance hydroxy- or amino-substitutedalkyl or benzyl. In a further embodiment, R^(a) and R^(b) areindependently benzyl or substituted alkyl. In a further embodiment,R^(y) is selected from the group consisting of alkyl and hydroxyalkyl.In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH. In certain embodimentsaccording to this paragraph, R² and R³ are each hydrogen, R^(a) ishydrogen, R^(b) is —CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In one embodiment, R^(y) is alkyl or hydroxyalkyl. In one embodiment,R^(y) is methyl, tert-butyl, hydroxy-tert-butyl or hydroxyethyl. Incertain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In one embodiment, R^(a) and R^(b) are each independently hydrogen,alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,aminocarbonylalkyl, alkoxycarbonylalkyl, aryl or arylalkyl, wherein thealkyl groups can be further substituted with one or more substitutents.In one embodiment, at least one of R^(a) or R^(b) is other thanhydrogen. In one embodiment, R^(a) and R^(b) are each independentlyhydrogen, methyl or benzyl.

In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH and R^(a) and R^(b) areeach independently hydrogen, methyl or benzyl. In certain embodiments,R^(y) is —C(CH₃)₂CH₂OH and R^(a) is hydrogen and R^(b) is benzyl.

In another embodiment, the compound provided herein is a compound offormula:

wherein R¹ and R^(y) are as defined in formula IIa or IIb. In oneembodiment, R^(y) is alkyl or hydroxyalkyl. In one embodiment, R^(y) ismethyl, tert-butyl, hydroxy-tert-butyl or hydroxyethyl. In oneembodiment, R^(y) is —C(CH₃)₂CH₂OH.

In certain embodiments according to formula XVa or XVb, R^(y) issubstituted alkyl, e.g. hydroxyalkyl or aminoalkyl. In anotherembodiment, R^(y) is —OR^(c), —C(R^(c))₃ or —NHR^(c) where each R^(c) isindependently alkyl, substituted alkyl, aryl or substituted aryl, forinstance hydroxy- or amino-substituted alkyl or aryl. In a furtherembodiment, R^(y) is selected from the group consisting of alkyl andhydroxyalkyl. In certain embodiments, R^(y) is —C(CH₃)₂CH₂OH.

In another embodiment, the compound provided herein is a compound offormula:

Wherein:

R¹ is an anti-cancer drug, such as a nucleoside or nucleosidederivative; and

R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, arylalkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; and

wherein in one embodiment, one of R^(a) and R^(b) is H and the other isalkyl optionally substituted with aryl, benzyl, or heteroaryl, eachoptionally substituted.

In certain embodiments according to formula XVIa or XVIb, R^(a) andR^(b) are independently hydrogen, alkyl, substituted alkyl, benzyl orsubstituted benzyl, for instance hydroxy- or amino-substituted alkyl orbenzyl. In another embodiment, R^(a) and R^(b) are independentlyhydrogen, alkyl, substituted alkyl, benzyl or substituted benzyl, forinstance hydroxy- or amino-substituted alkyl or benzyl. In a furtherembodiment, R^(a) and R^(b) are independently benzyl or substitutedalkyl.

In another embodiment, the compound provided herein is a compound offormula:

wherein R¹ is a drug such as an anti-cancer drug.

Exemplary anti-cancer drugs (R¹'s) that can be derivatized as describedherein, for example via a free hydroxyl group, or after adding ahydroxylated linker, are:

Name Structure Aclarubicin

Decitabine

Daunorubicin

5-azacytidine

Doxorubicin

Epirubicin

Estramustine

Etoposide

Fludarabine

Neplanocin A

Tezacitabine([(E)-2′-deoxy-2′-(fluoromethylene)cytidine(FMdC)])

Troxacitabine((-)-2′-Deoxy-3′-oxacytidine)

Vinblastin

Vincristin

Vindesin

Teniposide

NK-611

Camptothecin

Irinotecan

9-Aminocamptothecin

Topotecan

Paclitaxel

Azatoxin

Coformycin

Pirarubicin

Nelarabine

Losoxantrone

Fluxoridine

Mitomycin C

Erlotinib (Tarceva ®)

Thalidomide

Exemplary immunosupressant drugs that can be derivatized as describedherein are:

Mitoxantrone

CombretastatinA-4

Mycophenolicacid

Pentostatin

In certain embodiments, R¹ is an anti-cancer drug such as aclarubicin,decitabine, daunorubicin, dihydro-5-azacytidine, doxorubicin,epirubicin, estramustin, etoposide, fludarabine, 7-hydroxychlorpromazin,neplanocin A, podophyllotoxin, tezacitabine, troxacitabine, vinblastin,vincristin, vindesin, etoposide, teniposide, NK-611, camptothecin,irinotecan, 9-aminocamptothecin, GG-211, topotecan, paclitaxel,azatoxin, coformycin, pirarubicin and losoxantrone. In a particularembodiment, the anti-cancer drug is camptothecin or azotoxin.

In another embodiment, R¹ is a purine nucleoside analog (see, e.g.,Robak et al., Curr. Med. Chem. 2006, 13, 3165-3189). R¹ is, for example,a cytotoxic agent such as fludarabine(9-β-D-arabinofuranosyl-2-fluoradenine), cladribine(2-chloro-2′-deoxyadenosine, CldA), pentostatin (2′-deoxycoformycin,DCF), clofarabine (CAFdA), nelabarine, immucillin H (BCX-1777,forodesine) or 8-chloroadenosine (8-Cl-Ado).

The anti-cancer drug also can be (2′S)-2′-deoxy-2′-C-methylcytidine(SMDC), 1-(2-deoxy-2-methylene-β-D-erythro-pentofuranosyl)cytosine(DMDC), 1-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)cytosine(CNDAC) or 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd). See,e.g., Matsuda et al., Cancer Sci, 2004, 95:105-111.

In one embodiment, R¹ is an immunosuppressant, such as combretastatinA-4, mycophenolic acid, pentostatin or mitoxantrone.

The anti-cancer drug can be derivatized to include the phosphoroamidateor phosphonoamidate at, e.g., a free OH or free carboxy group.

In another embodiment, R¹ is (−)-2′-Deoxy-3′-oxacytidine (BCH-4556,Troxacitabine):

In another embodiment, R¹ is tezacitabine(2′-fluoromethylene-2′-deoxycytidine). In another embodiment, R¹ is a5′-aza-pyrimidine, such as 5′-aza-cytidine, 5′-azadeoxycytidine(decytabine), or fazarabine.

In certain embodiments, R¹ is a 2′-deoxy-2′-methylidenepyrimidinenucleoside compound, disclosed, e.g. in U.S. Pat. No. 5,401,726, such as2′-deoxy-2′-methylidene-5-fluorocytidine,2′-deoxy-2′-methylidene-5-chlorocytidine,2′-deoxy-2′-methylidene-5-bromocytidine,2′-deoxy-2′-methylidene-5-iodocytidine,2′-deoxy-2′-methylidene-5-methylcytidine,2′-deoxy-2′-methylidene-5-ethylcytidine,2′-deoxy-2′-methylidene-5-ethyluridine,2′-deoxy-2′-methylidene-5-ethynyluridine or2′-deoxy-2′-methylidene-5-fluorocytidine-5′-phosphoric acid. In oneembodiment, R¹ is 5-fluorouracil.

R¹ also can be a pyrido[2,3-D]pyrimidine or pyrimido[4,5-D]pyrimidinenucleoside as described in U.S. Pat. No. 7,081,449, such as4-amino-5-oxo-8-(4-C-hydroxymethyl-β-D-ribofuranosyl)pyrido-[2,3d]pyrimidine;4-amino-5-oxo-8-(5(R)—C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-amino-5-oxo-8-(5(R)—C-allyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-amino-5-oxo-8-(5(R,S)—C-ethynyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(5(R,S)—C-vinyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-amino-5-oxo-8-(β-L-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-amino-5-oxo-8-(4-C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-amino-5-oxo-8-(4-C-ethyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-amino-5-oxo-8-(5(R,S)—C-ethyl-β-D-ribofuranosyl)pyrido-[2,3d]pyrimidine;4-amino-5-oxo-8-(5(R)—C-propyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;or 4-amino-5-oxo-8-(2-deoxy-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine.In a particular embodiment, R¹ is4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine.

Other anti-cancer drugs include: dichloroacetal, Nexavar (sorafenib),cimetidine, adriamycin, Cytoxan (cyclophosphamide), methotrexate,vincristine, and 6-mercaptopurine.

In another embodiment, the anti-cancer drug is selected from2′,3-dideoxyinosine (ddl), or 2,3-didehydro-3-deoxythymidine (d4T). See,e.g., WO/2006/125166.

In one embodiment, R¹ is an anti-inflammatory drug, such as acorticosteroid or a non-steroidal anti-inflammatory drugs (NSAID) thatcan be derivatized to include the phosphoroamidate or phosphonoamidateat, e.g., a free OH or free carboxy group.

Exemplary corticosteroid drugs suitable for use herein are providedbelow:

Exemplary NSAID drugs suitable for use herein are provided below:

Sodium salicylate Salicylsalicylic acid

In certain embodiments of the compounds of Formula IIa below:

the moiety:

is derived from a drug that is an acyclic nucleoside phosphonate, i.e.:

Thus, compounds of Formula IIa, in one embodiment, are phosphonoamidatesof an acyclic nucleoside phosphonate that have potential anti-canceractivity, such as (S)-9-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine(HPMPC, cidofovir), (S)-9-{3-hydroxy-2-(phosphonomethoxy)-propyl]adenine((S)-HPMPA), phosphonomethoxyethylguanine (PMEG),phosphonomethoxyethyl-adenine (PMEA, adefovir),phosphonomethoxy-propyladenine (PMPA, tenofovir), acyclovir, gancicloviror penciclovir. See e.g., WO/2006/125166 and De Clercq et al., AntiviralResearch, Volume 75, Issue 1, July 2007, Pages 1-13.

Embodiments for Delivery of Thyroid Hormone Receptor Effectors

In certain embodiments, the following phosphoroamidate andphosphonoamidate formulas and compounds are provided, which optionallyact as thyroid hormone receptor effectors:

wherein

each R, if present, is independently alkyl, halogen or hydroxyl;

X, if present, is CH₂, O or S;

R^(y) is alkyl, alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl,cycloalkenyl, amino, aminoalkyl, heterocyclyl or heteroaryl, alloptionally substituted;

R^(a) and R^(b) are selected as follows:

i) R^(a) and R^(b) are each independently hydrogen, alkyl, carboxyalkyl,hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, heteroaryl orheterocyclyl, all optionally substituted; or

ii) R^(a) and R^(b) together with the nitrogen atom on which they aresubstituted form a 3-7 membered heterocyclic or heteroaryl ring.

In certain embodiments, the compound provided herein has formulaselected from IIIa, IIIb, IVa, IVb, Va, Vb, VIa, VIb, VIIa, VIIb, VIIIaor VIIIb, wherein

each R, if present, is independently alkyl, halogen or hydroxyl;

X, if present, is CH₂, O or S;

R^(y), if present, is optionally substituted alkyl, wherein thesubstituted alkyl is optionally hydroxyalkyl or aminoalkyl, e.g.,—C(CH₃)₂CH₂OH; and

R^(a) and R^(b), if present, are independently hydrogen; unsubstitutedalkyl; or alkyl substituted with aryl, amino, amido, hydroxyl, alkoxy,aminoalkyl, hydroxyalkyl, aryl, or heteroaryl, each optionallysubstituted; wherein, in one embodiment, R^(a) and R^(b) areindependently H or a benzyl that is optionally substituted, for example,with hydroxy or amino; and

-   -   wherein, in another embodiment, if present, R^(a) is hydrogen,        R^(b) is —CH₂—C₆H₅ and R^(y) is —C(CH₃)₂CH₂OH.

In certain embodiments, the compound provided herein has a formulaselected from:

wherein

R^(x) and R^(z) are each independently hydrogen or alkyl;

R^(w) is alkyl;

X¹ is O or S;

R^(y) is optionally substituted alkyl, wherein the substituents whenpresent are selected from hydroxy and amino;

R^(a) and R^(b) are each independently hydrogen or optionallysubstituted alkyl; where the substituents when present are selected fromone or more, in one embodiment, one, two or three groups selected fromaryl, amino, amido, hydroxyl, alkoxy, aryl and heteroaryl, eachoptionally substituted with hydroxy or amino.

In certain embodiments, the compound provided herein is selected from:

In certain embodiments, R^(x) and R^(z) are each hydrogen. In certainembodiments, R^(w) is alkyl. In certain embodiments, R^(w) is isopropyl.In certain embodiments, R^(y) is optionally substituted alkyl, whereinthe substituents when present are selected from hydroxy and amino. Incertain embodiments, R^(y) is —C(CH₃)₂CH₂OH. In certain embodiments,R^(a) and R^(b) are each independently hydrogen or optionallysubstituted alkyl; where the substituents when present are selected fromone or more, in one embodiment, one, two or three groups selected fromaryl, amino, amido, hydroxyl, alkoxy, aryl and heteroaryl, eachoptionally substituted with hydroxy or amino. In certain embodiments,R^(a) is hydrogen and R^(b) is benzyl.

In certain embodiments, R^(a) is hydrogen, R^(b) is —CH₂—C₆H₅ and R^(y)is —C(CH₃)₂CH₂OH.

In one embodiment, the thyroid receptor effector compound has formula:

In certain embodiments, the compound or Formula selected from IIIa or b,IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b is derived from aphosphonate compound useful for inhibiting gluconeogenesis, optionallyby inhibiting the enzyme fructose 1,6-bisphosphatase (FBPase).

In certain embodiments, the compound or Formula selected from IXa or b,X a or b, XIa or b, XIIa or b, XIIIa or b, XIVa or b and XVIIIa or b isderived from a compound useful for inhibiting gluconeogenesis,optionally by inhibiting the enzyme fructose 1,6-bisphosphatase(FBPase).

In certain embodiments, the compound or Formula selected from IIIa or b,IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b is a phosphonicacid-containing compound that binds to a thyroid receptor in the liver,and is optionally an agonist, antagonist, partial agonist or partialantagonist of T3. Inhibition of gluconeogenesis can result in bloodglucose lowering in diabetic subjects. Such compounds can exhibitenhanced pharmacokinetics including oral bioavailability and liver druglevels.

In certain embodiments, provided is a method of treatment of a subjectin need thereof, the method comprising administering to the subject aphosphoroamidate and phosphonoamidate compound or formula selected fromIIIa or b, IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b, IXa orb, X a or b, XIa or b, XIIa or b, XIIIa or b, XIVa or b and XVIIIa or bor a pharmaceutically acceptable salt, enantiomer, ester or prodrugthereof thereof, in an amount effective for one or more of thefollowing:

reducing plasma lipid levels, lowering cholesterol levels, reducingtriglyceride levels, or increasing the ratio of HDL to LDL;

lowering blood glucose levels;

treating hyperlipidemia or hypercholesterolemia;

treating obesity, reducing fat content, treating fatty liver, reducingweight or preventing weight gain;

treating atherosclerosis, coronary heart disease, heart failure,nephrotic syndrome, or chronic renal failure;

lowering blood glucose levels, treating diabetes, impaired glucosetolerance, metabolic syndrome x, insulin resistance or hyperinsulinemia;

increasing levels of genes associated with gluconeogenesis;

decreasing hepatic glycogen levels or maintaining or improving glycemiccontrol;

amelioration of hyperinsulinemia and/or decrease of glucose levels indiabetic subjects at doses that optionally do not affect cardiacfunction, e.g., heart rate, force of systolic contraction, duration ofdiastolic relaxation, vascular tone, or heart weight;

treating thyroid disease, thyroid cancer, depression, glaucoma, cardiacarrhythmias, heart failure, or osteoporosis;

increasing mitochondrial biogenesis, or increasing expression of PGC-1,AMP activated protein kinase or nuclear respiratory factor;

inhibiting hepatic gluconeogenesis; or

modulating expression of certain genes in the liver resulting in effectson lipids (e.g., cholesterol), glucose, lipoproteins, and triglycerides,or modulation of T3-responsive genes.

In certain embodiments, the compounds do not affect thyroid function,thyroid production of circulating iodinated thyronines such as T3 andT4, and/or the ratio of T3 to T4.

In certain embodiments, provided herein is a method for treatment ofliver fibrosis or inflammation by administering a compound providedherein.

Also provided are pharmaceutical compositions comprising the compounds,e.g., in a dosage unit suitable for administration, e.g., oraladministration.

Optically Active Compounds

It is appreciated that compounds provided herein have several chiralcenters and may exist in and be isolated in optically active and racemicforms. Some compounds may exhibit polymorphism. It is to be understoodthat any racemic, optically-active, diastereomeric, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound provided herein,which possess the useful properties described herein is within the scopeof the invention. Techniques known in the art can be used to prepareoptically active forms (for example, by resolution of the racemic formby recrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase).

Examples of methods to obtain optically active materials are known inthe art, and include at least the following.

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.

Preparation of Compounds

The compounds provided herein can be prepared, isolated or obtained byany method apparent to those of skill in the art. Exemplary methods ofpreparation are described in detail in the examples below.

In certain embodiments, compounds provided herein can be prepared bycoupling alcohols and H-phosphonate monoesters as illustrated in thereaction scheme below:

wherein R⁷, R⁸, R⁹, R¹⁰ are each independently hydrogen, hydroxy, alkylor alkoxy. Any reactive function on R^(y), R⁷, R⁸, R⁹, R¹⁰ or on thebase should be protected during the coupling reaction. Any couplingagent known to one of skill in the art can be used. Exemplary couplingagents for use in the reaction include, but are not limited to HOBt(N-Hydroxybenzotriazole), HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate), DCC (N,N′-dicyclohexylcarbodiimide), BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate),PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophosphoniumhexafluorophosphate) and others known to one of skill in the art.

A general scheme for the synthesis of hydroxytBuSATEN-benzylphosphoramidate nucleoside derivatives represented by B isprovided in Schemes B1-B3 below, in which modifications of nucleosidesare made by way of example, but the methodology may be used for otheractive agents as well.

where R=H, Tr, MMTr or DMTr in case of reactive amine; R¹, R², R⁴, R⁶=H,alkyl or halo and R³/R⁵ are both H or isopropylidene.

In addition, certain nucleosides and analogs thereof and prodrugsthereof can be prepared according to methods known to one of skill inthe art. Exemplary nucleosides and analogs are described inInternational Publication No. WO 06/125166, contents of which are herebyincorporated by reference in their entireties.

The compounds of formula IIIa or b, IVa or b, Va or b, VIa or b, VIIa orb and VIII a or b can be prepared by methods described herein andmethods known to one of skill in the art, for example, see, Erion etal., Proc. Natl. Acad. Sci., 2007, 104, 15490-15495.

The compounds of formula IXa or b, X a or b, XIa or b, XIIa or b, XIIIaor b, XIVa or b and XVIIIa or b can be prepared by methods describedherein and methods known to one of skill in the art, for example, see,Dang et al., Discovery of Potent and SpecificFructose-1,6-Bisphosphatase Inhibitors and a Series oforally-Bioavailable Phosphoramidase-Sensitive Prodrugs for the Treatmentof Type 2 Diabetes, J. Am. Chem. Soc., 2007, Vol. 129, No. 50, pp.15491-502.

Assay Methods

Compounds can be assayed for accumulation in liver cells of a subjectaccording to any assay known to those of skill in the art. In certainembodiments, a compound can be administered to the subject, and a livercell of the subject can be assayed for the compound or a derivativethereof.

In one embodiment, a phosphoroamidate or phosphonoamidate nucleosidecompound is administered to cells, such as liver cells, in vivo or invitro, and the levels delivered intracellularly are measured, toindicate delivery of the compound in the cell.

Assays for other activities, including anti-cancer activity can be doneas described in the art. Suitable in vitro assays can be used topreliminarily evaluate the efficacy of a compound in inhibiting growthof cancer cells. The compound can further be examined for its efficacyin treating cancer by in vivo assays known to those of skill in the art.For example, it can be administered to an animal (e.g., a mouse model)having cancer and its therapeutic effect can then assessed. Based on theresults, an appropriate dosage range and administration route can alsobe determined. Exemplary assays are described in the paragraphs below.

Anti-Cancer Activity

Compounds provided herein can be shown to inhibit tumor cellproliferation, cell transformation and tumorigenesis in vitro and invivo using a variety of assays known in the art, or described herein.Such assays can use cells of a cancer cell line, or cells from apatient. Many assays well-known in the art can be used to assess suchsurvival and/or growth; for example, cell proliferation can be assayedby measuring (³H)-thymidine incorporation, by direct cell count, bydetecting changes in transcription, translation or activity of knowngenes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers(Rb, cdc2, cyclin A, D1, D2, D3, E, etc.). The levels of such proteinand mRNA and activity can be determined by any method well known in theart. For example, protein can be quantitated by known immunodiagnosticmethods such as Western blotting or immunoprecipitation usingcommercially available antibodies (for example, many cell cycle markerantibodies are available from Santa Cruz Biotechnology, Inc., SantaCruz, Calif.). mRNA can be quantitated by methods that are well knownand routine in the art, for example, by Northern analysis, RNaseprotection, and the polymerase chain reaction in connection with thereverse transcription. Cell viability can be assessed by usingtrypan-blue staining or other cell death or viability markers known inthe art. Differentiation can be assessed, for example, visually based onchanges in morphology, etc.

Cell proliferation analysis can be performed using a variety oftechniques known in the art, including but not limited to the following:

As one example, bromodeoxyuridine (BRDU) incorporation may be used as anassay to identify proliferating cells. The BRDU assay identifies a cellpopulation undergoing DNA synthesis by incorporation of BRDU into newlysynthesized DNA. Newly synthesized DNA can then be detected using ananti-BRDU antibody (see Hoshino et al., 1986, Int. J. Cancer 38, 369;Campana et al., 1988, J. Immunol. Meth. 107, 79).

Cell proliferation can also be examined using (3H)-thymidineincorporation (see e.g., Chen, J., 1996, Oncogene 13:1395-403; Jeoung,J., 1995, J. Biol. Chem. 270:18367-73). This assay allows forquantitative characterization of S-phase DNA synthesis. In this assay,cells synthesizing DNA incorporate (3H)-thymidine into newly synthesizedDNA. Incorporation can then be measured using standard techniques in theart such as by counting of radioisotope in a Scintillation counter (e.g.Beckman LS 3800 Liquid Scintillation Counter).

Detection of proliferating cell nuclear antigen (PCNA) can also be usedto measure cell proliferation. PCNA is a 36 kilodalton protein whoseexpression is elevated in proliferating cells, particularly in early G1and S phases of the cell cycle and therefore can serve as a marker forproliferating cells. Positive cells are identified by immunostainingusing an anti-PCNA antibody (see Li et al., 1996, Curr. Biol. 6:189-199;Vassilev et al., 1995, J. Cell Sci. 108:1205-15).

Cell proliferation can be measured by counting samples of a cellpopulation over time (e.g. daily cell counts). Cells may be countedusing a hemacytometer and light microscopy (e.g. HyLite hemacytometer,Hausser Scientific). Cell number may be plotted against time in order toobtain a growth curve for the population of interest. In a preferredembodiment, cells counted by this method are first mixed with the dyeTrypan-blue, such that living cells exclude the dye, and are counted asviable members of the population.

DNA content and/or mitotic index of the cells can be measured, forexample, based on the DNA ploidy value of the cell. For example, cellsin the GI phase of the cell cycle generally contain a 2N DNA ploidyvalue. Cells in which DNA has been replicated but have not progressedthrough mitosis (e.g. cells in S-phase) exhibit a ploidy value higherthan 2N and up to 4N DNA content. Ploidy value and cell-cycle kineticscan be further measured using propidum iodide assay (see e.g. Turner,T., et al., 1998, Prostate 34:175-81). Alternatively, the DNA ploidy canbe determined by quantitation of DNA Feulgen staining (which binds toDNA in a stoichiometric manner) on a computerizedmicrodensitometrystaining system (see e.g., Bacus, S., 1989, Am. J.Pathol. 135:783-92). In an another embodiment, DNA content can beanalyzed by preparation of a chromosomal spread (Zabalou, S., 1994,Hereditas. 120:127-40; Pardue, 1994, Meth. Cell Biol. 44:333-351).

The expression of cell-cycle proteins (e.g., CycA, CycB, CycE, CycD,cdc2, Cdk4/6, Rb, p21, p27, etc.) provide information relating to theproliferative state of a cell or population of cells. For example,identification in an anti-proliferation signaling pathway can beindicated by the induction of p21. Increased levels of p21 expression incells results in delayed entry into G1 of the cell cycle (Harper et al.,1993, Cell 75:805-816; Li et al., 1996, Curr. Biol. 6:189-199). p21induction can be identified by immunostaining using a specific anti-p21antibody available commercially (e.g. Santa Cruz Biotechnology, Inc.,Santa Cruz, Calif.). Similarly, cell-cycle proteins may be examined byWestern blot analysis using commercially available antibodies. Inanother embodiment, cell populations are synchronized prior to detectionof a cell cycle protein. Cell cycle proteins can also be detected byFACS (fluorescence-activated cell sorter) analysis using antibodiesagainst the protein of interest.

Detection of changes in length of the cell cycle or speed of cell cyclecan also be used to measure inhibition of cell proliferation by thecompounds provided herein. In one embodiment the length of the cellcycle is determined by the doubling time of a population of cells (e.g.,using cells contacted or not contacted with one or more compoundsidentified using the pharmacophores of the present invention). Inanother embodiment, FACS analysis is used to analyze the phase of cellcycle progression, or purify G1, S, and G2/M fractions (see e.g., Delia,D. et al., 1997, Oncogene 14:2137-47).

The compounds useful in the methods of the present invention can also bedemonstrated to inhibit cell transformation (or progression to malignantphenotype) in vitro. In this embodiment, cells with a transformed cellphenotype are contacted with one or more compounds of the presentinvention, and examined for change in characteristics associated with atransformed phenotype (a set of in vitro characteristics associated witha tumorigenic ability in vivo), for example, but not limited to, colonyformation in soft agar, a more rounded cell morphology, loosersubstratum attachment, loss of contact inhibition, loss of anchoragedependence, release of proteases such as plasminogen activator,increased sugar transport, decreased serum requirement, or expression offetal antigens, etc. (see Luria et al., 1978, General Virology, 3d Ed.,John Wiley & Sons, New York, pp. 436-446).

Loss of invasiveness or decreased adhesion may also be used todemonstrate the anti-cancer effects of the compounds useful in themethods of the present invention. For example, a critical aspect of theformation of a metastatic cancer is the ability of a precancerous orcancerous cell to detach from primary site of disease and establish anovel colony of growth at a secondary site. The ability of a cell toinvade peripheral sites is reflective of a potential for a cancerousstate. Loss of invasiveness may be measured by a variety of techniquesknown in the art including, for example, induction ofE-cadherin-mediated cell-cell adhesion. Such E-cadherin-mediatedadhesion can result in phenotypic reversion and loss of invasiveness(Hordijk et al., 1997, Science 278:1464-66).

Loss of invasiveness may further be examined by inhibition of cellmigration. A variety of 2-dimensional and 3-dimensional cellularmatrices are commercially available (Calbiochem-Novabiochem Corp. SanDiego, Calif.). Cell migration across or into a matrix may be examinedby microscopy, time-lapsed photography or videography, or by any methodin the art allowing measurement of cellular migration. In a relatedembodiment, loss of invasiveness is examined by response to hepatocytegrowth factor (HGF). HGF-induced cell scattering is correlated withinvasiveness of cells such as Madin-Darby canine kidney (MDCK) cells.This assay identifies a cell population that has lost cell scatteringactivity in response to HGF (Hordijk et al., 1997, Science 278:1464-66).

Alternatively, loss of invasiveness may be measured by cell migrationthrough a chemotaxis chamber (Neuroprobe/Precision Biochemicals Inc.,Vancouver, BC). In such assay, a chemo-attractant agent is incubated onone side of the chamber (e.g., the bottom chamber) and cells are platedon a filter separating the opposite side (e.g., the top chamber). Inorder for cells to pass from the top chamber to the bottom chamber, thecells must actively migrate through small pores in the filter.Checkerboard analysis of the number of cells that have migrated may thenbe correlated with invasiveness (see e.g., Ohnishi, T., 1993, Biochem.Biophys. Res. Commun. 193:518-25).

The compounds provided herein can also be demonstrated to inhibit tumorformation in vivo. A number of animal models of hyperproliferativedisorders, including tumorigenesis and metastatic spread, are known inthe art (see Table 317-1, Chapter 317, “Principals of Neoplasia,” inHarrison's Principals of Internal Medicine, 13th Edition, Isselbacher etal., eds., McGraw-Hill, New York, p. 1814, and Lovejoy et al., 1997, J.Pathol. 181:130-135).

For example, a compound provided herein can be administered to a testanimal, preferably a test animal predisposed to develop a type of tumor,and the test animal subsequently examined for decreased incidence oftumor formation in comparison with controls not administered thecompound identified using the pharmacophores of the present invention.Alternatively, a compound useful in the methods of the present inventioncan be administered to test animals having tumors (e.g., animals inwhich tumors have been induced by introduction of malignant, neoplastic,or transformed cells, or by administration of a carcinogen) andsubsequently examining the tumors in the test animals for tumorregression in comparison to controls that were not administered thecompound.

Thyroid Receptor Binding Activity

Thyroid receptor binding activity that can serve as a mechanism fortreatment of diseases sensitive thereto can be tested using assaysavailable in the art. Thyroid hormones (TH) are synthesized in thethyroid in response to thyroid stimulating hormone (TSH), which issecreted by the pituitary gland in response to various stimulants.Thyroid hormones are iodinated O-aryl tyrosine analogues excreted intothe circulation primarily as 3,3′,5,5′-tetraiodothyronine (T4). T4 israpidly deiodinated in local tissues by thyroxine 5′-deiodinase to3,3′,5′-triiodothyronine (T3), which is the most potent TH. Most of thecirculating T4 and T3 is eliminated through the liver.

THs have profound physiological effects in animals and humans.Hyper-thyroidism is associated with increased body temperature, generalnervousness, weight loss despite increased appetite, muscle weakness andfatigue, increased bone resorption and enhanced calcification, and avariety of cardiovascular changes, including increased heart rate,increased stroke volume, increased cardiac index, cardiac hypertrophy,decreased peripheral vascular resistance, and increased pulse pressure.Hypothyroidism is generally associated with the opposite effects.

The biological activity of THs is mediated largely through thyroidhormone receptors (TRs). TRs belong to the nuclear receptor superfamily,which, along with its common partner, the retinoid X receptor, formheterodimers that act as ligand-inducible transcription factors.

The most widely recognized effects of THs are an increase in metabolicrate, oxygen consumption and heat production. T3 treatment increasesoxygen consumption in isolated perfused liver and isolated hepatocytes.(Oh et al., J. Nutr. 125(1):112-24 (1995); Oh et al., Proc. Soc. Exp.Biol. Med. 207(3): 260-7 (1994))

THs also stimulate metabolism of cholesterol to bile acids.Hyperthyroidism leads to decreased plasma cholesterol levels, which islikely due to increased hepatic LDL receptor expression. Hypothyroidismis a well-established cause of hypercholesterolemia and elevated serumLDL. L-T3 is known to lower plasma cholesterol levels. In addition, THsare known to affect levels of other lipoproteins linked toatherosclerosis. THs stimulate apo AI and the secretion of apo AI in HDLwhile reducing apo B100. Accordingly, one would expect T3 and T3mimetics to inhibit the atherosclerotic process in the cholesterol fedanimal.

THs simultaneously increase de novo fatty acid synthesis and oxidationthrough effects on enzymes such as ACC, FAS, and spot-14. THs increasecirculating free fatty acids (FFA) levels in part by increasingproduction of FFAs from adipose tissue via TH-induced lipolysis. Inaddition, THs increase mitochondrial enzyme levels involved in FFAoxidation, e.g., carnitine palmitoyltransferase 1 (CPT-1) and enzymesinvolved in energy storage and consumption.

The liver represents a major target organ of THs. Microarray analysis ofhepatic gene expression from livers of hypothyroid mice and mice treatedwith T3 showed changes in mRNA levels for 55 genes (14 positivelyregulated and 41 negatively regulated) (Feng et al., Mol. Endocrinol.14(7): 947-55 (2000). Others have estimated that approximately 8% of thehepatic genes are regulated by T3. Many of these genes are important toboth fatty acid and cholesterol synthesis and metabolism. T3 is alsoknown to have other effects in liver, including effects on carbohydratesthrough increased glycogenolysis and gluconeogenesis and decreasedinsulin action.

TH has been used as an antiobesity drug for over 50 years. In the 1940sTH was used alone, whereas in the 1950s it was used in combination withdiuretics and in the 1960s in combination with amphetamines. Treatinghypothyroidism patients with T3 leads to a decrease in body weight formost patients. T3 and T3 mimetics are thought to inhibit atherosclerosisby modulating the levels of certain lipoproteins known to be independentrisk factors or potential risk factors of atherosclerosis, including lowdensity lipoprotein (LDL)-cholesterol, high density lipoprotein(HDL)-cholesterol, apoAI, which is a major apoprotein constituent ofhigh density lipoprotein (HDL) particles and lipoprotein (a) or Lp(a).

Hyperthyroidism worsens glycemic control in type 2 diabetics. TH therapyis reported to stimulate hepatic gluconeogenesis. Enzymes specific togluconeogenesis and important for controlling the pathway and itsphysiological role of producing glucose are known to be influenced by THtherapy. Phosphoenolpyruvate carboxykinase (PEPCK) is upregulated by TH(Park et al, J. Biol. Chem. 274:211 (1999)) whereas others have foundthat glucose 6-phosphatase is upregulated (Feng et al., Mol. Endocrinol.14:947 (2000)). TH therapy is also associated with reduced glycogenlevels. TH therapy results in improved non insulin stimulated andinsulin stimulated glucose utilization and decreased insulin resistancein the muscle of ob/ob mice. (Oh et al., J. Nutr. 125:125 (1995)).

Thus, thyromimetics potentially can be used to modulate cholesterollevels, to treat obesity, and other metabolic disorders especially withreduced undesirable effects.

Studies can be used to determine the affinity of T3 and variousthyromimetics for human thyroid hormone receptors TRα1 and TRβ1, andtheir resulting efficacy in related disorders. Binding of compounds toeither the TRα1 or TRβ₁ receptors can be performed by means ofscintillation proximity assays (SPA). The SPA assay, a common methodused for the quantitation of receptor-ligand equilibria, makes use ofspecial beads coated with a scintillant and a capture molecule, copper,which binds to the histidine-tagged α or β receptor. When labeled T3 ismixed with receptor and the SPA beads, radioactive counts are observedonly when the complex of protein and radiolabeled ligand is captured onthe surface of the bead. Displacement curves are generated with labeledT3 and increasing concentrations of unlabeled thyromimetics of interest.Subacute studies can be used in ZDF Rats (Charles River Laboratory) todemonstrate an improved therapeutic index for T3 Mimetics.

Subacute studies also can be conducted in cholesterol-fed rats. Thecholesterol-fed rat is an animal model of hypercholesterolemia generatedby feeding the animals a diet with high cholesterol content. The purposeof these studies is to evaluate the effects of compounds on serumcholesterol (an efficacy parameter) and on heart weight and heart mGPDHactivity (potential toxicity parameters). Compounds can be administered,e.g., IP, e.g., once-a-day for seven days.

Microsome/primary hepatocyte stability studies can be conducted usingmethods available in the art. Prodrug activation in rat liver microsomescan be conducted to determine the kinetics of activation of prodrugs ofthyromimetics in microsomal preparations. Microsomes may contain P450enzyme that may activate a prodrug. The Km, Vmax, and intrinsicclearance values determined are measures of prodrug affinity for themicrosomal enzymes, the rate at which the prodrug is activated, and thecatalytic efficiency with which the prodrug is activated, respectively.Prodrugs also can be tested for conversion to their respective parentcompounds by human liver S9. The S9 fraction is a fraction that containsboth cytosolic and microsomal protein. Uptake and activation of prodrugin isolated rat hepatocytes also can be conducted using methods known inthe art. Oral bioavailability and liver distribution following oraladministration also can be measured using methods available in the art.

Oxygen consumption studies can be conducted. Thermogenesis is ameasurement of energy consumption. Compounds that increase thermogenesisare likely to increase caloric expenditure and thereby cause body weightloss and its associated benefits to metabolic status (e.g., insulinsensitivity). Thermogenesis is assessed in subcellular fractions ofvarious tissues, isolated cells, whole tissues, or in whole animalsusing changes in oxygen consumption as the endpoint. Oxygen is used upwhen calories are burned by various metabolic processes.

Mitochondrial thermogenesis is measured polarographically with aClark-type oxygen electrode using mitochondria isolated from varioustissues, including liver. Mitochondria are isolated by differentialcentrifugation. State 3 respiration or cytochrome c oxidase activity aremeasured in isolated mitochondria. (Iossa, S, FEBS Letters, 544: 133-7(2003)). Oxygen consumption rates are measured in isolated hepatocytesusing a portable Clark-type oxygen electrode placed in the hepatocytemedium. Hepatocytes are isolated from liver using a two-step collagenaseperfusion (Berry, M. N., Friend, D. S. J. Cell Biol. 43: 506-520 (1969))as modified by Groen (Groen, A. K. et al., Eur J. Biochem 122: 87-93(1982)). Non-parenchymal cells are removed using a Percoll gradient andthe cells are resuspended in tissue culture medium in a spinner flask.The oxygen consumption of the cells is measured over time once thesystem is sealed.

Oxygen consumption also can be measured in isolated perfused liver(Fernandez, V., Toxicol Lett. 69:205-10 (1993)). Liver is perfused insitu and oxygen consumption is calculated by measuring the differencebetween the oxygen saturation of the inflow buffer and the outflowbuffer maintained at a constant flow. Whole animal oxygen consumptioncan be measured using an indirect calorimeter (Oxymax, ColumbusInstruments, Columbus, Ohio). Animals are removed from their cages andplaced in the chambers. The resting oxygen consumption is measured inanimals during periods of inactivity as measured by activity monitors.The oxygen consumption is calculated based on the flow through thechamber and the difference in oxygen partial pressures at the inflow andoutlet ports. Carbon dioxide efflux is also measured in parallel using aCO2 electrode.

Tissue distribution and the pharmacokinetics of compounds can beassessed following IP or oral administration to normal rats.

Studies can be conducted to evaluate the effects of a T3 mimetic onserum cholesterol and TSH levels, hepatic and cardiac gene expressionand enzyme activities, heart weight, and clinical chemistry parametersusing methods available in the art. In one embodiment, rats are madehypercholesterolemic by maintenance on a diet containing 1.5%cholesterol and 0.5% cholic acid for at least 2 weeks prior toinitiation of treatment. Plasma cholesterol values are assessed prior toand following treatment and the effects of compound are expressed as apercentage change from the pre-dose cholesterol levels. Totalcholesterol is analyzed using a commercially available enzymatic kit(Sigma Diagnostics, St. Louis, Mo.).

Effects of T3 mimetic compounds (and prodrugs thereof) in vivo onglucose can be measured in ZDF rats. T3 and T3 mimetic mediated myosinheavy chain gene transcription in the heart can be measured. An RT-PCRassay as disclosed in: Sara Danzi, Kaie Ojamaa, and Irwin Klein Am JPhysiol Heart Circ Physiol 284: H2255-H2262, 2003 is used to study boththe time course and the mechanism for the triiodothyronine (T3)-inducedtranscription of the α- and β-myosin heavy chain (MHC) genes in vivo onthe basis of the quantity of specific heterogeneous nuclear RNA (hnRNA).The temporal relationship of changes in transcriptional activity to theamount of α-MHC mRNA and the coordinated regulation of transcription ofmore than one gene in response to T3 and T3 mimetics are demonstrated.Analysis of a time course of T3 and T3 mimetics that are not liverspecific show mediated induction of α-MHC hnRNA and repression of β-MHChnRNA, whereas no significant affect is observed with compounds at dosesthat are therapeutically useful.

The effect of T3 on cardiovascular function (heart rate, inotropicstate, and aortic pressure) can be studied in the Sprague Dawley (SD)rat model using assay methods known in the art.

Thus, various assays known in the art can be used to assay for thyroidhormone agonist and its accompanying therapeutic activity and toestablish appropriate dosages.

Methods of Use

The phosphoroamidate and phosphonoamidate compounds of a variety oftherapeutic agents can be formed using methods available in the art andthose disclosed herein. Such compounds can be used in some embodimentsto enhance delivery of the drug to the liver. In one embodiment, thecompound comprises a S-acyl-2-thioethyl phosphoroamidate orS-acyl-2-thioethyl phosphonoamidate, e.g., a S-pivaloyl-2-thioethylphosphoroamidate or S-hydroxypivaloyl-2-thioethyl phosphonoamidatederivative. Therapeutic agents that can be derivatized tophosphoroamidate or phosphonoamidate compound form include a therapeuticagent such as an anti-cancer agent or anti-diabetic agent that includes,or has been derivatized to include a reactive group for attachment ofthe phosphoroamidate or phosphonoamidate moiety.

Cancer Treatment Methods

In one embodiment, therapeutic agents for the treatment of liver cancercan be derivatized to form a phosphoroamidate or phosphonoamidatecompound as described herein, and used for the treatment of livercancers. Liver cancers that can be treated include benign tumors,malignant tumors, hemangioma, hepatic adenomas, focal nodularhyperplasia, hepatocellular carcinoma, fibrolamellar carcinoma,cholangiocarcinomas, bile duct cancers, and other primary and metastaticcancers of the liver.

Exemplary therapeutic agents include anti-cancer agents having one ormore hydroxy groups that can be derivatized as compounds describedherein by removal of a hydrogen from one of the hydroxy groups.Exemplary anti-cancer agents include, but are not limited toaclarubicin, decitabine, daunorubicin, dihydro-5-azacytidine,doxorubicin, epirubicin, estramustin, etoposide, fludarabine,7-hydroxychlorpromazin, neplanocin A, podophyllotoxin, tezacitabine,troxacitabine, vinblastin, vincristin, vindesin, etoposide, teniposide,NK-611, camptothecin, irinotecan, 9-aminocamptothecin, GG-211,topotecan, paclitaxel, azatoxin, coformycin, pirarubicin, nelarabine andlosoxantrone. Anti-cancer agents known in the art and described hereincan be derivatized to form a phosphoramidate or phosphonoamidatecompound as described herein. Immunosuppressants, such as combretastatinA-4, mycophenolic, pentostatin, or mitoxantrone, also can be derivatizedto form a phosphoramidate or phosphonoamidate compound as describedherein.

Such compounds can optionally be used in combination with anotheranti-cancer agent that is optionally in prodrug form.

Methods of Treating Metabolic Diseases

In certain embodiments, the compounds provided herein are useful inmethods for inhibiting gluconeogenesis, optionally by inhibiting theenzyme fructose 1,6-bisphosphatase (FBPase).

In certain embodiments, the compounds provided herein are useful inmethods for inhibiting gluconeogenesis.

In certain embodiments, the compounds provided herein are useful inmethods for treatment of metabolic diseases. In certain embodiments, thecompounds provided herein are useful in methods for:

reducing plasma lipid levels, lowering cholesterol levels, reducingtriglyceride levels, or increasing the ratio of HDL to LDL;

lowering blood glucose levels;

treating hyperlipidemia or hypercholesterolemia;

treating obesity, reducing fat content, treating fatty liver, reducingweight or preventing weight gain;

treating atherosclerosis, coronary heart disease, heart failure,nephrotic syndrome, or chronic renal failure;

lowering blood glucose levels, treating diabetes, impaired glucosetolerance, metabolic syndrome x, insulin resistance or hyperinsulinemia;

increasing levels of genes associated with gluconeogenesis;

decreasing hepatic glycogen levels or maintaining or improving glycemiccontrol;

amelioration of hyperinsulinemia and/or decrease of glucose levels indiabetic subjects at doses that optionally do not affect cardiacfunction, e.g., heart rate, force of systolic contraction, duration ofdiastolic relaxation, vascular tone, or heart weight;

treating thyroid disease, thyroid cancer, depression, glaucoma, cardiacarrhythmias, heart failure, or osteoporosis;

increasing mitochondrial biogenesis, or increasing expression of PGC-1,AMP activated protein kinase or nuclear respiratory factor;

inhibiting hepatic gluconeogenesis; or

modulating expression of certain genes in the liver resulting in effectson lipids (e.g., cholesterol), glucose, lipoproteins, and triglycerides,or modulation of T3-responsive genes.

In certain embodiments, the compounds provided herein are useful inmethods for lowering blood glucose levels, treating diabetes, impairedglucose tolerance, metabolic syndrome x, insulin resistance orhyperinsulinemia.

Second Agents Useful in the Methods

In certain embodiments, the compounds and compositions provided hereinare useful in methods of treatment of a liver disorder, that comprisesfurther administration of a second agent effective for the treatment ofthe disorder, such as liver cancer in a subject in need thereof. Thesecond agent can be any agent known to those of skill in the art to beeffective for the treatment of the disorder, including those currentlyapproved by the FDA.

In certain embodiments, a compound provided herein is administered incombination with one second agent. In further embodiments, a secondagent is administered in combination with two second agents. In stillfurther embodiments, a second agent is administered in combination withtwo or more second agents.

As used herein, the term “in combination” includes the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).The use of the term “in combination” does not restrict the order inwhich therapies (e.g., prophylactic and/or therapeutic agents) areadministered to a subject with a disorder. A first therapy (e.g., aprophylactic or therapeutic agent such as a compound provided herein)can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to a subject with a disorder.

As used herein, the term “synergistic” includes a combination of acompound provided herein and another therapy (e.g., a prophylactic ortherapeutic agent) which has been or is currently being used to prevent,manage or treat a disorder, which is more effective than the additiveeffects of the therapies. A synergistic effect of a combination oftherapies (e.g., a combination of prophylactic or therapeutic agents)permits the use of lower dosages of one or more of the therapies and/orless frequent administration of said therapies to a subject with adisorder. The ability to utilize lower dosages of a therapy (e.g., aprophylactic or therapeutic agent) and/or to administer said therapyless frequently reduces the toxicity associated with the administrationof said therapy to a subject without reducing the efficacy of saidtherapy in the prevention or treatment of a disorder). In addition, asynergistic effect can result in improved efficacy of agents in theprevention or treatment of a disorder. Finally, a synergistic effect ofa combination of therapies (e.g., a combination of prophylactic ortherapeutic agents) may avoid or reduce adverse or unwanted side effectsassociated with the use of either therapy alone.

In certain embodiments, the active compounds provided herein can beadministered in combination or alternation with another therapeuticagent, for example an anti-cancer agent. In certain embodiments, theactive compounds provided herein can be administered in combination oralternation with second agents useful in treating metabolic disorderssuch as diabetes, obesity, atherosclerosis, heart disease, metabolicsyndrome x, nephrotic syndrome, thyroid disease, and symptoms associatedtherewith. In combination therapy, effective dosages of two or moreagents are administered together, whereas in alternation orsequential-step therapy, an effective dosage of each agent isadministered serially or sequentially. The dosages given will depend onabsorption, inactivation and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens and schedules should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions.

The second agent can be one of the agents disclosed herein. In certainembodiments, contemplated additional pharmaceutically active substancesinclude drugs commonly used as chemotherapy for treatment of cancer andimmune modulator substances. For example, chemotherapeutic agentsinclude anti-metabolites (e.g., Pentostatin®), DNA polymerase inhibitors(e.g, Gemzar®), RNA polymerase inhibitors (e.g., ECyd®), platinumderivatives (e.g., Paraplatin®), anti-estrogens (e.g., Nolvadex®),Taxanes (e.g., Taxotere®), GnRH analogs (e.g., Lupron®), DNA polymeraseinhibitors (e.g., Gemzar®), topoisomerase inhibitors (e.g., Hycamptin®),biphosphonates (e.g., Aredia®), somatostatins (e.g., Sandostatin®),nucleoside analogs (e.g., Ribavirin®), and IMPDH-inhibitors (e.g.,Tiazofurin®). Contemplated immunomodulatory substances include cytokines(e.g., interferon α and γ, IL2, IL4, IL6, IL8, IL10, and IL12),cytokinins (e.g., kinetin), and chemokines (e.g., MIP-1).

In certain embodiments, the second agents for use in combination withthe compounds provided herein include other agents useful in thetreatment, prevention, suppression or amelioration of the diseases orconditions for which compounds provided herein are useful, such astreating metabolic diseases, including diabetes, obesity,atherosclerosis, heart disease, metabolic syndrome x, nephroticsyndrome, thyroid disease, and symptoms associated therewith. Suchsecond agents include, but are not limited to: sulfonylureas, forexample, glibenclamide (DAONIL®), glimepiride (AMARYL®), glipizide(GLUCOTROL or MINODIAB), glyburide (MICRONASE®), tolbutamide (ORINASE®),acetohexamide (DYMELOR®), tolazamide (TOLINSE®) and chlorpropamide,(DIABINESE®); insulin and insulin mimetics; biguanides such as metformin(GLUCOPHAGE®); α-glucosidase inhibitors including acarbose (PRECOSE®)and miglitol (GLYSET®); meglitinides, for example, nateglinide(STARLIX®) and repaglinide (PRANDIN®); thiozolidinediones, for example,ciglitazone, englitazone, rosiglitazone (AVANDIA®), pioglitazone(ACTOS®) and troglitazone (REZULIN®); incretin mimetics such asexenatide (BYETTA™); cholesterol lowering agents such as HMG-CoAreductase inhibitors (e.g., lovastatin, simvastatin, pravastatin,fluvastatin, atorvastatin and other statins), bile acid sequestrants(e.g., cholestyramine and colestipol), vitamin B₃ (also known asnicotinic acid, or niacin), vitamin B₆ (pyridoxine), vitamin B₁₂(cyanocobalamin), fibric acid derivatives (e.g., gemfibrozil,clofibrate, fenofibrate and benzafibrate), probucol, and inhibitors ofcholesterol absorption (e.g., beta-sitosterol and acylCoA-cholesterolacyltransferase (ACAT) inhibitors such as melinamide), HMG-CoA synthaseinhibitors, squalene epoxidase inhibitors and squalene synthetaseinhibitors; antithrombotic agents, such as thrombolytic agents (e.g.,streptokinase, alteplase, anistreplase and reteplase), heparin, hirudinand warfarin derivatives, β-blockers (e.g., atenolol), β-adrenergicagonists (e.g., isoproterenol) and ACE inhibitors and vasodilators(e.g., sodium nitroprusside, nicardipine hydrochloride, nitroglycerinand enaloprilat).

Pharmaceutical Compositions and Methods of Administration

Phosphoroamidate and phosphonoamidate compounds of a variety oftherapeutic agents can be formulated into pharmaceutical compositionsusing methods available in the art and those disclosed herein. Suchcompounds can be used in some embodiments to enhance delivery of thedrug to the liver. In one embodiment, the compound comprises aS-acyl-2-thioethyl phosphoroamidate or S-acyl-2-thioethylphosphonoamidate, e.g., a S-pivaloyl-2-thioethyl phosphoroamidate orS-hydroxypivaloyl-2-thioethyl phosphonoamidate derivative. In certainembodiments, therapeutic agents that can be derivatized tophosphoroamidate or phosphonoamidate compound form include anyanti-cancer agent that includes, or has been derivatized to include areactive group for attachment of the phosphoroamidate orphosphonoamidate moiety, including but not limited to nucleosides andnucleoside analogues including acyclic nucleosides. In certainembodiments, therapeutic agents that can be derivatized tophosphoroamidate or phosphonoamidate compound form include any thyroidharmone receptor effector that includes, or has been derivatized toinclude a reactive group for attachment of the phosphoroamidate orphosphonoamidate moiety. Any of the phosphoroamidate or phosphonoamidatecompounds disclosed herein can be provided in the appropriatepharmaceutical composition and be administered by a suitable route ofadministration.

The methods provided herein encompass administering pharmaceuticalcompositions containing at least one compound as described herein,including a compound of general formula I, Ia, IIb, IIIa, IVa, IXa orIXb if appropriate in the salt form, either used alone or in the form ofa combination with one or more compatible and pharmaceuticallyacceptable carriers, such as diluents or adjuvants, or with othertherapeutic agents, such as another anti-cancer or anti-diabetic agent.

In certain embodiments, the second agent can be formulated or packagedwith the compound provided herein. Of course, the second agent will onlybe formulated with the compound provided herein when, according to thejudgment of those of skill in the art, such co-formulation should notinterfere with the activity of either agent or the method ofadministration. In certain embodiments, the compound provided herein andthe second agent are formulated separately. They can be packagedtogether, or packaged separately, for the convenience of thepractitioner of skill in the art.

In clinical practice the active agents provided herein may beadministered by any conventional route, in particular orally,parenterally, rectally or by inhalation (e.g. in the form of aerosols).In certain embodiments, the compound provided herein is administeredorally.

Use may be made, as solid compositions for oral administration, oftablets, pills, hard gelatin capsules, powders or granules. In thesecompositions, the active product is mixed with one or more inertdiluents or adjuvants, such as sucrose, lactose or starch.

These compositions can comprise substances other than diluents, forexample a lubricant, such as magnesium stearate, or a coating intendedfor controlled release.

Use may be made, as liquid compositions for oral administration, ofsolutions which are pharmaceutically acceptable, suspensions, emulsions,syrups and elixirs containing inert diluents, such as water or liquidparaffin. These compositions can also comprise substances other thandiluents, for example wetting, sweetening or flavoring products.

The compositions for parenteral administration can be emulsions orsterile solutions. Use may be made, as solvent or vehicle, of propyleneglycol, a polyethylene glycol, vegetable oils, in particular olive oil,or injectable organic esters, for example ethyl oleate. Thesecompositions can also contain adjuvants, in particular wetting,isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization can be carried out in several ways, for example using abacteriological filter, by radiation or by heating. They can also beprepared in the form of sterile solid compositions which can bedissolved at the time of use in sterile water or any other injectablesterile medium.

The compositions for rectal administration are suppositories or rectalcapsules which contain, in addition to the active principle, excipientssuch as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

The compositions can also be aerosols. For use in the form of liquidaerosols, the compositions can be stable sterile solutions or solidcompositions dissolved at the time of use in apyrogenic sterile water,in saline or any other pharmaceutically acceptable vehicle. For use inthe form of dry aerosols intended to be directly inhaled, the activeprinciple is finely divided and combined with a water-soluble soliddiluent or vehicle, for example dextran, mannitol or lactose.

In one embodiment, a composition provided herein is a pharmaceuticalcomposition or a single unit dosage form. Pharmaceutical compositionsand single unit dosage forms provided herein comprise a prophylacticallyor therapeutically effective amount of one or more prophylactic ortherapeutic agents (e.g., a compound provided herein, or otherprophylactic or therapeutic agent), and a typically one or morepharmaceutically acceptable carriers or excipients. In a specificembodiment and in this context, the term “pharmaceutically acceptable”includes approval by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” includes a diluent, adjuvant (e.g., Freund'sadjuvant (complete and incomplete)), excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water can be used as a carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a subjectand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions provided herein can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose freecompositions comprise an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose free dosage forms comprise an activeingredient, microcrystalline cellulose, pre gelatinized starch, andmagnesium stearate.

Further encompassed herein are anhydrous pharmaceutical compositions anddosage forms comprising active ingredients, since water can facilitatethe degradation of some compounds. For example, the addition of water(e.g., 5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long term storage in order to determine characteristics suchas shelf life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, NY, N.Y., 1995, pp. 379 80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine can be anhydrousif substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions can be packaged using materials known to prevent exposureto water such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

Further provided are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and single unit dosage forms can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of aprophylactic or therapeutic agent, in certain embodiments, in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject. The formulation shouldsuit the mode of administration. In a certain embodiment, thepharmaceutical compositions or single unit dosage forms are sterile andin suitable form for administration to a subject, for example, an animalsubject, such as a mammalian subject, for example, a human subject.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral, e.g., intravenous,intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal,sublingual, inhalation, intranasal, transdermal, topical, transmucosal,intra-tumoral, intra-synovial and rectal administration. In a specificembodiment, the composition is formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenous,subcutaneous, intramuscular, oral, intranasal or topical administrationto human beings. In an embodiment, a pharmaceutical composition isformulated in accordance with routine procedures for subcutaneousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocamne to ease pain at the site of theinjection.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a subject,including suspensions (e.g., aqueous or non aqueous liquid suspensions,oil in water emulsions, or a water in oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a subject; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a subject.

The composition, shape, and type of dosage forms provided herein willtypically vary depending on their use. For example, a dosage form usedin the initial treatment of liver cancer may contain larger amounts ofone or more of the active ingredients it comprises than a dosage formused in the maintenance treatment of the same liver cancer. Similarly, aparenteral dosage form may contain smaller amounts of one or more of theactive ingredients it comprises than an oral dosage form used to treatthe same disease or disorder. These and other ways in which specificdosage forms encompassed herein will vary from one another will bereadily apparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Typical dosage forms comprise a compound provided herein, or apharmaceutically acceptable salt, solvate or hydrate thereof lie withinthe range of from about 0.1 mg to about 1000 mg per day, given as asingle once-a-day dose in the morning or as divided doses throughout theday taken with food. Particular dosage forms can have about 0.1, 0.2,0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100,200, 250, 500 or 1000 mg of the active compound.

Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be presented as discrete dosage forms, such as, but are not limitedto, tablets (e.g., chewable tablets), caplets, capsules, and liquids(e.g., flavored syrups). Such dosage forms contain predetermined amountsof active ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

In certain embodiments, the oral dosage forms are solid and preparedunder anhydrous conditions with anhydrous ingredients, as described indetail in the sections above. However, the scope of the compositionsprovided herein extends beyond anhydrous, solid oral dosage forms. Assuch, further forms are described herein.

Typical oral dosage forms are prepared by combining the activeingredient(s) in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms include,but are not limited to, binders, fillers, disintegrants, and lubricants.Binders suitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch,hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions is typically presentin from about 50 to about 99 weight percent of the pharmaceuticalcomposition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC581, AVICEL PH 105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL PH 103™ and Starch 1500LM.

Disintegrants are used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients should be used to form solid oral dosage forms.The amount of disintegrant used varies based upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. Typical pharmaceutical compositions comprise from about 0.5 toabout 15 weight percent of disintegrant, specifically from about 1 toabout 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, pre gelatinized starch, other starches, clays, otheralgins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, for example, a syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulatedaerosol of synthetic silica (marketed by Degussa Co. of Plano, Tex.),CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. ofBoston, Mass.), and mixtures thereof. If used at all, lubricants aretypically used in an amount of less than about 1 weight percent of thepharmaceutical compositions or dosage forms into which they areincorporated.

Delayed Release Dosage Forms

Active ingredients such as the compounds provided herein can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358;6,699,500 each of which is incorporated herein by reference. Such dosageforms can be used to provide slow or controlled release of one or moreactive ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the active ingredients provided herein.Thus encompassed herein are single unit dosage forms suitable for oraladministration such as, but not limited to, tablets, capsules, gelcaps,and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased subject compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

In certain embodiments, the drug may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(see, Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in a subject at anappropriate site determined by a practitioner of skill, i.e., thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)). The active ingredient can be dispersedin a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient then diffuses through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive ingredient in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the needs of the subject.

Parenteral Dosage Forms

In one embodiment, provided are parenteral dosage forms. Parenteraldosage forms can be administered to subjects by various routesincluding, but not limited to, subcutaneous, intravenous (includingbolus injection), intramuscular, and intraarterial. Because theiradministration typically bypasses subjects' natural defenses againstcontaminants, parenteral dosage forms are typically, sterile or capableof being sterilized prior to administration to a subject. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms.

Transdermal, Topical & Mucosal Dosage Forms

Also provided are transdermal, topical, and mucosal dosage forms.Transdermal, topical, and mucosal dosage forms include, but are notlimited to, ophthalmic solutions, sprays, aerosols, creams, lotions,ointments, gels, solutions, emulsions, suspensions, or other forms knownto one of skill in the art. See, e.g., Remington's PharmaceuticalSciences, 16^(th), 18th and 20^(th) eds., Mack Publishing, Easton Pa.(1980, 1990 & 2000); and Introduction to Pharmaceutical Dosage Forms,4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes or as oral gels. Further, transdermal dosage forms include“reservoir type” or “matrix type” patches, which can be applied to theskin and worn for a specific period of time to permit the penetration ofa desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed herein are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3diol, isopropyl myristate, isopropyl palmitate, mineral oil, andmixtures thereof to form lotions, tinctures, creams, emulsions, gels orointments, which are non toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18th and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 & 2000).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients provided. For example, penetration enhancers canbe used to assist in delivering the active ingredients to the tissue.Suitable penetration enhancers include, but are not limited to: acetone;various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkylsulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethylformamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery enhancing orpenetration enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which heconsiders most appropriate according to a preventive or curativetreatment and according to the age, weight, stage of the disease, forexample, cancer and other factors specific to the subject to be treated.In certain embodiments, doses are from about 1 to about 1000 mg per dayfor an adult, or from about 5 to about 250 mg per day or from about 10to 50 mg per day for an adult. In certain embodiments, doses are fromabout 5 to about 400 mg per day or 25 to 200 mg per day per adult. Incertain embodiments, dose rates of from about 50 to about 500 mg per dayare also contemplated.

In further aspects, provided are methods of treating or preventing livercancer in a subject by administering, to a subject in need thereof, aneffective amount of a compound provided herein, or a pharmaceuticallyacceptable salt thereof. In other aspects, provided are methods oftreating or preventing metabolic diseases in a subject by administering,to a Subject in need thereof, an effective amount of a compound providedherein, or a pharmaceutically acceptable salt thereof. The amount of thecompound or composition which will be effective in the prevention ortreatment of a disorder or one or more symptoms thereof will vary withthe nature and severity of the disease or condition, and the route bywhich the active ingredient is administered. The frequency and dosagewill also vary according to factors specific for each subject dependingon the specific therapy (e.g., therapeutic or prophylactic agents)administered, the severity of the disorder, disease, or condition, theroute of administration, as well as age, body, weight, response, and thepast medical history of the subject. Effective doses may be extrapolatedfrom dose-response curves derived from in vitro or animal model testsystems.

In certain embodiments, exemplary doses of a composition includemilligram or microgram amounts of the active compound per kilogram ofsubject or sample weight (e.g., about 10 micrograms per kilogram toabout 50 milligrams per kilogram, about 100 micrograms per kilogram toabout 25 milligrams per kilogram, or about 100 microgram per kilogram toabout 10 milligrams per kilogram). For compositions provided herein, incertain embodiments, the dosage administered to a subject is 0.140 mg/kgto 3 mg/kg of the subject's body weight, based on weight of the activecompound. In certain embodiments, the dosage administered to a subjectis between 0.20 mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50mg/kg of the subject's body weight.

In certain embodiments, the recommended daily dose range of acomposition provided herein for the conditions described herein liewithin the range of from about 0.1 mg to about 1000 mg per day, given asa single once-a-day dose or as divided doses throughout a day. In oneembodiment, the daily dose is administered twice daily in equallydivided doses. In certain embodiments, a daily dose range should be fromabout 10 mg to about 200 mg per day, in other embodiments, between about10 mg and about 150 mg per day, in further embodiments, between about 25and about 100 mg per day. It may be necessary to use dosages of theactive ingredient outside the ranges disclosed herein in some cases, aswill be apparent to those of ordinary skill in the art. Furthermore, itis noted that the clinician or treating physician will know how and whento interrupt, adjust, or terminate therapy in conjunction with subjectresponse.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to prevent,manage, treat or ameliorate such disorders, but insufficient to cause,or sufficient to reduce, adverse effects associated with the compositionprovided herein are also encompassed by the above described dosageamounts and dose frequency schedules. Further, when a subject isadministered multiple dosages of a composition provided herein, not allof the dosages need be the same. For example, the dosage administered tothe subject may be increased to improve the prophylactic or therapeuticeffect of the composition or it may be decreased to reduce one or moreside effects that a particular subject is experiencing.

In certain embodiment, the dosage of the composition provided herein,based on weight of the active compound, administered to prevent, treat,manage, or ameliorate a disorder, or one or more symptoms thereof in asubject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. Inanother embodiment, the dosage of the composition or a compositionprovided herein administered to prevent, treat, manage, or ameliorate adisorder, or one or more symptoms thereof in a subject is a unit dose of0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg,0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg,1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In certain embodiments, treatment or prevention can be initiated withone or more loading doses of a compound or composition provided hereinfollowed by one or more maintenance doses. In such embodiments, theloading dose can be, for instance, about 60 to about 400 mg per day, orabout 100 to about 200 mg per day for one day to five weeks. The loadingdose can be followed by one or more maintenance doses. In certainembodiments, each maintenance does is, independently, about from about10 mg to about 200 mg per day, between about 25 mg and about 150 mg perday, or between about 25 and about 80 mg per day. Maintenance doses canbe administered daily and can be administered as single doses, or asdivided doses.

In certain embodiments, a dose of a compound or composition providedherein can be administered to achieve a steady-state concentration ofthe active ingredient in blood or serum of the subject. The steady-stateconcentration can be determined by measurement according to techniquesavailable to those of skill or can be based on the physicalcharacteristics of the subject such as height, weight and age. Incertain embodiments, a sufficient amount of a compound or compositionprovided herein is administered to achieve a steady-state concentrationin blood or serum of the subject of from about 300 to about 4000 ng/mL,from about 400 to about 1600 ng/mL, or from about 600 to about 1200ng/mL. In some embodiments, loading doses can be administered to achievesteady-state blood or serum concentrations of about 1200 to about 8000ng/mL, or about 2000 to about 4000 ng/mL for one to five days. Incertain embodiments, maintenance doses can be administered to achieve asteady-state concentration in blood or serum of the subject of fromabout 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, orfrom about 600 to about 1200 ng/mL.

In certain embodiments, administration of the same composition may berepeated and the administrations may be separated by at least 1 day, 2days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or 6 months. In other embodiments, administration of thesame prophylactic or therapeutic agent may be repeated and theadministration may be separated by at least at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months.

In certain aspects, provided herein are unit dosages comprising acompound, or a pharmaceutically acceptable salt thereof, in a formsuitable for administration. Such forms are described in detail above.In certain embodiments, the unit dosage comprises 1 to 1000 mg, 5 to 250mg or 10 to 50 mg active ingredient. In particular embodiments, the unitdosages comprise about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mgactive ingredient. Such unit dosages can be prepared according totechniques familiar to those of skill in the art.

The dosages of the second agents are to be used in the combinationtherapies provided herein. In certain embodiments, dosages lower thanthose which have been or are currently being used to prevent or treatthe diseases described herein, for example, liver cancer and diabetes,are used in the combination therapies provided herein. The recommendeddosages of second agents can be obtained from the knowledge of those ofskill. For those second agents that are approved for clinical use,recommended dosages are described in, for example, Hardman et al., eds.,1996, Goodman & Gilman's The Pharmacological Basis Of Basis OfTherapeutics 9th Ed, Mc-Graw-Hill, New York; Physician's Desk Reference(PDR) 57^(th) Ed., 2003, Medical Economics Co., Inc., Montvale, N.J.,which are incorporated herein by reference in its entirety.

In various embodiments, the therapies (e.g., a compound provided hereinand the second agent) are administered less than 5 minutes apart, lessthan 30 minutes apart, less than 1 hour apart, 1 hour apart, at about 1hour apart, at about 1 to about 2 hours apart, at about 2 hours to about3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In variousembodiments, the therapies are administered no more than 24 hours apartor no more than 48 hours apart. In certain embodiments, two or moretherapies are administered within the same patient visit. In otherembodiments, the compound provided herein and the second agent areadministered concurrently.

In certain embodiments, a compound provided herein and a second agentare administered to a patient, for example, a mammal, such as a human,in a sequence and within a time interval such that the compound providedherein can act together with the other agent to provide an increasedbenefit than if they were administered otherwise. For example, thesecond active agent can be administered at the same time or sequentiallyin any order at different points in time; however, if not administeredat the same time, they should be administered sufficiently close in timeso as to provide the desired therapeutic or prophylactic effect. In oneembodiment, the compound provided herein and the second active agentexert their effect at times which overlap. Each second active agent canbe administered separately, in any appropriate form and by any suitableroute. In other embodiments, the compound provided herein isadministered before, concurrently or after administration of the secondactive agent.

In other embodiments, the compound provided herein and the second agentare administered at about 2 to 4 days apart, at about 4 to 6 days apart,at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart.

In certain embodiments, the compound provided herein and the secondagent are cyclically administered to a patient. Cycling therapy involvesthe administration of a first agent (e.g., a first prophylactic ortherapeutic agents) for a period of time, followed by the administrationof a second agent and/or third agent (e.g., a second and/or thirdprophylactic or therapeutic agents) for a period of time and repeatingthis sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improve theefficacy of the treatment.

In certain embodiments, the compound provided herein and the secondactive agent are administered in a cycle of less than about 3 weeks,about once every two weeks, about once every 10 days or about once everyweek. One cycle can comprise the administration of a compound providedherein and the second agent by infusion over about 90 minutes everycycle, about 1 hour every cycle, about 45 minutes every cycle. Eachcycle can comprise at least 1 week of rest, at least 2 weeks of rest, atleast 3 weeks of rest. The number of cycles administered is from about 1to about 12 cycles, more typically from about 2 to about 10 cycles, andmore typically from about 2 to about 8 cycles.

In other embodiments, courses of treatment are administered concurrentlyto a patient, i.e., individual doses of the second agent areadministered separately yet within a time interval such that thecompound provided herein can work together with the second active agent.For example, one component can be administered once per week incombination with the other components that can be administered onceevery two weeks or once every three weeks. In other words, the dosingregimens are carried out concurrently even if the therapeutics are notadministered simultaneously or during the same day.

The second agent can act additively or synergistically with the compoundprovided herein. In one embodiment, the compound provided herein isadministered concurrently with one or more second agents in the samepharmaceutical composition. In another embodiment, a compound providedherein is administered concurrently with one or more second agents inseparate pharmaceutical compositions. In still another embodiment, acompound provided herein is administered prior to or subsequent toadministration of a second agent. Also contemplated are administrationof a compound provided herein and a second agent by the same ordifferent routes of administration, e.g., oral and parenteral. Incertain embodiments, when the compound provided herein is administeredconcurrently with a second agent that potentially produces adverse sideeffects including, but not limited to, toxicity, the second active agentcan advantageously be administered at a dose that falls below thethreshold that the adverse side effect is elicited.

Kits

Also provided are kits for use in methods of treatment of a liverdisorder such as cancer or metabolic diseases, such as diabetes,hyperlipidemia, atherosclerosis, and obesity. The kits can include acompound or composition provided herein, a second agent or composition,and instructions providing information to a health care providerregarding usage for treating the disorder. Instructions may be providedin printed form or in the form of an electronic medium such as a floppydisc, CD, or DVD, or in the form of a website address where suchinstructions may be obtained. A unit dose of a compound or compositionprovided herein, or a second agent or composition, can include a dosagesuch that when administered to a subject, a therapeutically orprophylactically effective plasma level of the compound or compositioncan be maintained in the subject for at least 1 days. In someembodiments, a compound or composition can be included as a sterileaqueous pharmaceutical composition or dry powder (e.g., lyophilized)composition.

In some embodiments, suitable packaging is provided. As used herein,“packaging” includes a solid matrix or material customarily used in asystem and capable of holding within fixed limits a compound providedherein and/or a second agent suitable for administration to a subject.Such materials include glass and plastic (e.g., polyethylene,polypropylene, and polycarbonate) bottles, vials, paper, plastic, andplastic-foil laminated envelopes and the like. If e-beam sterilizationtechniques are employed, the packaging should have sufficiently lowdensity to permit sterilization of the contents.

The following Examples illustrate the synthesis of representativecompounds provided herein. These examples are not intended, nor are theyto be construed, as limiting the scope of the claimed subject matter. Itwill be clear that the scope of claimed subject matter may be practicedotherwise than as particularly described herein. Numerous modificationsand variations of the subject matter are possible in view of theteachings herein and, therefore, are within the scope the claimedsubject matter.

EXAMPLES Example 1 Preparation of Hydroxy-tBuSATEN-benzylphosphoroamidate derivative A550 of L-2′,3′-dideoxyadenosineL-ddA

Synthetic Scheme:

Synthesis of Carboxylic Acid 2:

2,2-Dimethyl-3-hydroxypropanoic acid methyl ester (965 μL, 7.57 mmol)was added dropwise to a stirring solution of 4,4′-dimethoxytritylchloride (2.82 g, 8.33 mmol) in anhydrous pyridine (7.6 mL) at roomtemperature. The reaction mixture turned to a red solution quickly, thento an orange suspension (ca. 30 min), and this was left stirringovernight. The mixture was poured carefully over saturated aqueousNaHCO₃ solution (30 mL) and the product was extracted with Et₂O (3×20mL). The combined organic extracts were washed with brine (20 mL), dried(Na₂SO₄) and the volatiles were removed under reduced pressure. Theresulting oil was co-evaporated with toluene and the residue was quicklypurified by flash column chromatography (SiO₂, Ø=4 cm, H=20 cm) elutingwith 5→10→20→30% Et₂O in petroleum ether (40-60). Evaporation of thefractions (R_(f)=0.25, 30% Et₂O in petroleum ether (40-60)) affordedether 1 as a yellow oil (3.11 g, 95%). This compound (3.00 g, 6.91 mmol)was dissolved in THF (35 mL) and an aqueous solution of NaOH (10%, 3.5 gin 35 mL H₂O) was then added at room temperature. The solution turnedinstantly dark orange and this was stirred for 2 days. The medium wasthen carefully neutralized by dropwise addition of HCl (1M). The productwas extracted with Et₂O (4×50 mL) and the combined organic extracts werewashed with brine (50 mL), dried (Na₂SO₄) and the volatiles were removedunder reduced pressure. The crude yellow oil was quickly purified byflash column chromatography (SiO₂, Ø=2 cm, H=10 cm) eluting with 50%Et₂O in petroleum ether (40-60). Evaporation of the fractions affordedcarboxylic acid 2 as a white foam (2.23 g, 77%). R_(f)=0.50 (50% Et₂O inpetroleum ether (40-60)); ¹H-NMR (300 MHz, CDCl₃) 1.10 (s, 6H, 2×CH₃),3.06 (s, 2H, CH₂O), 3.65 (s, 6H, 2×OCH₃), 6.62-6.79 (m, 4H, PhCH),7.02-7.46 (stack, 8H, PhCH); ¹³C-NMR (75 MHz, CDCl₃) 22.6 (2×CH₃), 43.5(C(CH₃)₂), 55.1 (2×OCH₃), 85.9 (CPh₃), [125.3, 126.7, 127.7, 128.2,129.1, 130.0, 136.0, 144.9, 158.4 (Ph), some overlap], 182.2 (C═O).

Synthesis of Thioester 3:

1,1′-carbonyldiimidazole (830 mg, 5.12 mmol) was added to a stirringsolution of carboxylic acid 2 in anhydrous PhMe/DMF (2/1, v/v, 2.7 mL)at room temperature and the reaction mixture turned turbid instantly.After 30 min, the medium was diluted by adding anhydrous PhMe/DMF (93/7,v/v, 17 mL) and cooled to −10° C. 2-Mercaptoethanol (359 μL, 5.12 mmol)was then added dropwise and the solution was stirred for 1 h at thistemperature. The reaction mixture was diluted with H₂O (60 mL) and theproduct was extracted with Et₂O (3×15 mL). The combined organic extractswere washed with brine (15 mL), dried (Na₂SO₄) and the volatiles wereremoved under reduced pressure (bath temperature not exceeding 20° C.).The residue was purified by flash column chromatography (SiO₂, Ø=4 cm,H=15 cm, 1% Et₃N) eluting with 60-70% Et₂O in petroleum ether (40-60).Evaporation of the fractions afforded thioester 3 as a white syrup (1.74g, 92%) that solidified upon storage at 4° C. R_(f)=0.35 (70% Et₂O inpetroleum ether (40-60)); ¹H-NMR (300 MHz, CDCl₃) 1.16 (s, 6H, 2×CH₃),3.02 (t, J 6.0, 2H, CH₂S), 3.09 (s, 2H, CH₂O), 3.66 (t, J 6.0, 2H,CH₂OH), 3.72 (s, 6H, 2×OCH₃), 6.74-6.78 (m, 4H, PhCH), 7.09-7.36 (stack,8H, PhCH); ¹³C-NMR (75 MHz, CDCl₃) 22.9 (CH₃, 2×CH₃), 31.7 (CH₂, CH₂S),51.0 (quat. C, C(CH₃)₂), 55.2 (CH₃, 2×OCH₃), 61.9 (CH₂, CH₂OH), 70.0(CH₂, CH₂O), 85.8 (quat. C., CPh₃), [113.0 (CH, Ph), 126.7 (CH, Ph),127.7 (CH, Ph), 128.2 (CH, Ph), 130.1 (CH, Ph), some overlap], [135.9(quat. C, Ph), 144.8 (quat. C, Ph), 158.4 (quat. C, Ph), some overlap],205.0 (quat. C, C═O).

Synthesis of H-Phosphonate Monoester 4:

β-L-ddA (1.00 g, 4.25 mmol) was co-evaporated with anhydrous pyridine(3×10 mL) and then dissolved in anhydrous pyridine/DMF (1/1, v/v, 21mL). Diphenyl phosphite (5.76 mL, 29.8 mmol) was then added dropwise tothis solution at room temperature. The reaction mixture was stirred for20 min upon which a mixture of Et₃N/H₂O (1/1, v/v, 8.5 mL) was addeddropwise, and stirring was pursued for an additional 20 min. Thereaction mixture was concentrated under reduced pressure toapproximately 15-20 mL and this residue was directly purified by flashcolumn chromatography (SiO₂, Ø=4 cm, H=15 cm, 1% Et₃N) eluting slowlywith CH₂Cl₂ (150 mL) then 5% (200 mL) δ 10% (200 mL) δ 15% (300 mL) MeOHin CH₂Cl₂. Evaporation of the fractions afforded H-phosphonate monoester4 as a white foam (1.36 g, 80%) that could be kept for several weeks at4° C. R_(f)=0.10 (Et₃N/MeOH/CH₂Cl₂, Jan. 10, 1989); ¹H-NMR (300 MHz,CDCl₃) 1.21 (t, J 7.4, 9H, 3×NCH₂CH₃), 1.92-2.50 (stack, 4H, 2×2′-H,2×3′-H), 3.02 (q, J 7.4, 6H, 3×NCH₂CH₃), [3.96-4.03 and 4.18-4.30(stacks, 3H, 4′-H, 2×5′-H), 6.28 (m, 1′-H), 6.91 (d, J 623, 1H, P—H),7.05 (br s, 2H, NH₂), 8.21 (s, 1H), 8.54 (br s, 1H, OH), 8.57 (s, 1H).

Synthesis of Phosphoroamidate Diester 5:

H-Phosphonate monoester 4 (1.03 g, 2.57 mmol) and alcohol 3 (1.66 g,3.45 mmol) were co-evaporated with anhydrous pyridine (3×5 mL) and thendissolved in anhydrous pyridine (5 mL). PyBOP(1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate,1.60 g, 3.08 mmol) was then added in one portion and the reactionmixture was stirred for 15 min at room temperature. The solution waspoured over saturated aqueous NaHCO₃ solution (30 mL) and the productwas extracted with CH₂Cl₂ (4×15 mL). The combined organic extracts werewashed with brine (10 mL), dried (Na₂SO₄) and concentrated under reducedpressure to leave the corresponding H-phosphonate diester as a slightlyyellow oil (1.84 g, assuming 2.41 mmol). This was co-evaporated withanhydrous pyridine (3×5 mL; note: do not evaporate to dryness in orderto help further solubilization), and the residue was dissolved inanhydrous CCl₄ (24 mL). Benzylamine (791 μL, 7.23 mmol) was addeddropwise and the reaction mixture turned cloudy instantly (slight heatdevelopment was observed). The milky solution was stirred for 1 h atroom temperature and poured over saturated aqueous NaHCO₃ solution (30mL) and the product was extracted with CH₂Cl₂ (4×15 mL). The combinedorganic extracts were washed with brine (15 mL), dried (Na₂SO₄) andconcentrated under reduced pressure to afford phosphoroamidate diester 5as a yellow oil (2.00 g, assuming 2.31 mmol). This was used in the nextstep without any further purification. R_(f)=0.29 (4% MeOH in CH₂Cl₂);¹H-NMR (300 MHz, CDCl₃) 1.11 (s, 6H, 2×CH₃), 1.91-2.05 (m, 2H),2.31-2.59 (m, 2H), 3.06 (m, 2H, CH₂S), 3.08 (s, 2H, CH₂ODMTr), 3.69 (s,6H, 2×OCH₃), 3.83-4.28 (stacks, 7H, CH₂O, NCH₂Ph, 4′-H, 2×5′-H), 5.71(br s, 1H, NH), 6.18 (m, 1H, 1′-H), 6.69-6.80 (m, 4H, PhCH), 7.02-7.31(stack, 13H, PhCH), 7.90 (s, 1H), 8.01 (s, 1H), 8.23 (s, 2H, NH₂);¹³P-NMR (61 MHz, CDCl₃) 8.82, 8.99.

Synthesis of Hydroxy-tBuSATE N-benzylphosphoroamidate Derivative ofL-ddA:

Crude phosphoroamidate diester 5 (2.00 g, assuming 2.31 mmol) wasdissolved in dioxane/AcOH/H₂O (25/17/25, v/v/v, 462 mL) and the solutionwas stirred for 3 d at room temperature. Evaporation of the volatilesunder reduced pressure left a residue that was purified by flash columnchromatography (SiO₂, Ø=3 cm, H=15 cm) eluting with CH₂Cl₂ (100 mL) then2% (100 mL)→4% (100 mL)→6% (100 mL)→8% (150 mL) MeOH in CH₂Cl₂.Evaporation of the fractions left NM 204 as a white foam that wasdissolved in MeCN (5 mL). Upon addition of H₂O (5 mL), the solutionturned turbid and required sonication before lyophilization. Theresulting white powder was dried at room temperature (using P₂O₅ as adesiccant) under vacuum for 1 d. The title compound was obtained as ahighly hygroscopic white powder (1:1 mixture of diastereoisomers asjudged by ³¹P-NMR; 499 mg, 35% over 3 steps). [α]²⁰ _(D)=+4.2° (c 1.0,CHCl₃); R_(f)=0.29 (4% MeOH in CH₂Cl₂); ¹H-NMR (300 MHz, DMSO-d6) 1.10(s, 6H, 2×CH₃), 2.02-2.14 (m, 2H, 2×3′-H), 2.41-2.55 (m, 2H, 2×2′-H),3.01 (t, J 6.4, 2H, CH₂S), 3.43 (d, J 5.0, 2H, CH₂OH), 3.75-4.07 and4.18-4.29 (stacks, 7H, CH₂O, NCH₂Ph, 4′-H, 2×5′-H), 5.02 (t, J 5.0, 1H,OH), 5.62 (m, 1H, NH), 6.25 (t, J 5.1, 1H, 1′-H), 7.16-7.36 (stack, 7H,PhH, NH₂), 8.14 (s, 1H), 8.26 (s, 1H); ¹³C-NMR (75 MHz, DMSO-d6) 21.8(2×CH₃), 25.9 and 26.0 (CH₂, 3′-C), 28.2 and 28.3 (CH₂, CH₂S), 30.9 and31.0 (CH₂, 2′-C), 44.2 (CH₂, NCH₂Ph), 51.7 (quat. C, C(CH₃)₂), 63.7 and63.8 (CH₂, CH₂O), 66.8 (CH₂, m, 5′-C), 68.3 (CH₂, CH₂OH), 78.9 (CH, m,4′-C), 84.2 (CH, 1′-C), 118.9 (quat. C), [126.5 (CH, Ph), 127.2 (CH,Ph), 128.1 (CH, Ph), some overlap], 138.8 and 138.9 (CH), 140.5 and140.6 (quat. C), 148.9 (quat. C), 152.3 (CH), 155.0 (quat. C), 204.0(quat. C, C═O); ¹³P-NMR (61 MHz, DMSO-d6) 9.86, 9.95; m/z (FAB⁻) 563(2), 306 (76), 153 (100); HRMS 565.2034 ([M+H]⁺. C₂₄H₃₄O₆N₆PS requires565.1998); HPLC t_(R)=3.52 min (20% TEAC 20 mM in MeCN); UV (EtOH 95%)λ_(max)=259 (ε_(max) 15900), λ_(min)=224 (ε_(min) 7200).

Example 2 Preparation of Hydroxy-tBuSATE N-BenzylphosphoroamidateDerivative of 2′-C-methylcytidine

Synthesis of H-Phosphonate Monoester 5

Synthesis of Carboxylic Acid 3:

To a stirred solution of 2,2-dimethyl-3-hydroxypropanoic acid methylester (1, 15 ml, 117.6 mmol) in a mixture of anhydrous methylenechloride (590 ml) and triethylamine (23 ml), were addedtriphenylmethylene chloride (1.2 eq, 39.3 g) and 4-dimethylaminopyridine(0.1 eq, 1.44 g). The reaction mixture was left refluxing overnight. Themixture was poured carefully over a saturated aqueous NaHCO₃ solutionand the product was extracted with methylene chloride and washed withwater. The combined organic extracts were evaporated under reducedpressure to give crude compound 2 which will be used for the next stepwithout further purification. The resulting oil was dissolved in amixture of dioxan (350 ml) and an aqueous solution of NaOH (30%, 350ml). The heterogene mixture was refluxed for 16 hours. The reactionmixture was allowed to cool down to room temperature, the two phaseswere separated, and the organic phase carefully neutralized by dropwiseaddition of HCl (1M). The product was extracted with methylene chlorideand the organic phases were evaporated under reduced pressure. The crudeorange oil was recrystallized from methylene chloride to affordcarboxylic acid 3 as white crystals (92%). R_(f)=0.50 (70% diethyl etherin petroleum ether); ¹H-NMR (400 MHz, CDCl₃) 1.24 (s, 6H, 2×CH₃), 3.19(s, 2H, CH₂O), 7.2-7.5 (m, 15H, C₆H₅).

Synthesis of H-Phosphonate Monoester 5:

1,1′-carbonyldiimidazole (1.3 eq, 1.17 g) was added to a stirringsolution of carboxylic acid 3 (2 g, 5.56 mmol) in an anhydrous mixtureof toluene and dimethylformamide (2/1, v/v, 4.5 ml) at room temperature,and the reaction mixture turned turbid instantly. After 30 min, thereaction mixture was diluted with a mixture of toluene anddimethylformamide (93/7, v/v, 28 ml), cooled to −10° C., and2-mercaptoethanol (1.3 eq, 500 μL) was added. The solution was stirredfor 3 h at this temperature. The volatiles were removed under reducedpressure (bath temperature not exceeding 25° C.). The residue wasdissolved in methylene chloride and washed with water. The organicphases were combined, dried over sodium sulphate (Na₂SO₄), filtered andevaporated to dryness to give compound 4 as a yellow oil. This compoundwill be coevaporated with anhydrous pyridine and used for the next stepwithout further purification. R_(f)=0.71 (70% Et₂O in petroleum ether);¹H-NMR (400 MHz, CDCl₃) 1.20 (s, 6H, 2×CH₃), 3.05 (t, J=6.4 Hz, 2H,CH₂S), 3.15 (s, 2H, CH₂OTr), 3.69 (t, J=6.4 Hz, 2H, CH₂OH), 7.3-7.9 (m,15H, C₆H₅).

Phosphorus acid (10 eq, 4.1 g) was coevaporated two times with anhydrouspyridine, dissolved in that solvent (25 ml) and added to crude 4. Thereaction mixture was stirred at room temperature and a white precipitateappeared after few minutes. The reaction mixture was cooled down to 0°C. and pivaloyl chloride (5.5 eq, 3.4 ml) was added. The reactionmixture was allowed to warm to room temperature and stirred for 3 h. Thereaction was stopped by addition of a solution of triethylammoniumbicarbonate (TEAB 1M, 10 ml) and diluted with ethyl acetate (EtOAc).After extraction with EtOAc and TEAB 0.5M, the organic phases werecombined, dried over sodium sulphate, filtered and evaporated to dryness(bath temperature not exceeding 30° C.). The residue was purified byflash column chromatography eluting with 10% of methanol in methylenechloride+1% triethylamine. Evaporation of the fractions afforded theH-phosphonate monoester 5 as a white syrup (90%). R_(f)=0.25 (70% Et₂Oin petroleum ether); ¹H-NMR (400 MHz, CDCl₃) 1.17 (m, 2×CH₃+excess(CH₃CH₂)₃N), 2.9 (m, excess (CH₃CH₂)₃N), 3.12 (t, J=6.8 Hz, 2H, CH₂S),3.37 (s, 2H, CH₂OTr), 3.90 (m, 2H, CH₂OP), 7.2-7.6 (m, 15H, C₆H₅), 9.9(m, excess (CH₃CH₂)₃NH); ³¹P-NMR (161 MHz, CDCl₃) 3.85 (s).

Synthesis of Hydroxy-tBuSATE N-benzylphosphoroamidate derivative of2′-C-methylcytidine: The following two strategies were used for thesynthesis:

Strategy A Synthesis of the Protected Nucleoside 7

A mixture of 2′C-methylcytidine (NM107) (10 g, 39.0 mmol), triethylorthoformate (8.3 eq, 54 ml) and p-toluenesulfonic acid monohydrate (1eq, 7.4 g) in anhydrous acetone (650 ml), was refluxed overnight undernitrogen atmosphere. The reaction mixture was neutralized with anaqueous ammonia solution (26%) and the precipitate filtered. Thefiltrate was evaporated under reduced pressure and coevaporated withethanol. Purification of the crude mixture on silica gel columnchromatography (eluant: stepwise gradient [0-10%] of methanol inmethylene chloride) led to compound 6 as a pale-yellow solid (86%).R_(f)=0.30 (20% MeOH in methylene chloride), ¹H-NMR (400 MHz, DMSO-d₆)1.06 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 1.47 (s, 3H, CH₃), 3.6 (m, 2H,H-5′, H-5″), 4.1 (m, 1H, H-4′), 4.41 (d, 1H, H-3′, J=3.2 Hz), 5.16 (t,1H, OH-5′, J=4.0 Hz, D₂O exchangeable), 5.69 (d, 1H, H-5, J=8.0 Hz),6.04 (s, 1H, H-1′), 7.14-7.19 (bd, 2H, NH₂, D₂O exchangeable), 7.74 (d,1H, H-6, J=8.0 Hz); LC/MS Scan ES− 296 (M−H)⁻, Scan ES+ 298 (M+H)⁺,λ_(max)=280.7 nm.

Compound 6 (4.4 g, 14.8 mmol) was dissolved in anhydrous pyridine (74ml) and chlorotrimethylsilane (3 eq, 5.4 ml) was added. The reactionmixture was stirred at room temperature under nitrogen atmosphere for 2h, then 4,4′-dimethoxytrityl chloride (1.5 eq, 7.5 g) and4-dimethylaminopyridine (0.5 eq, 900 mg) were successively added. Thereaction mixture was stirred overnight at room temperature, thenquenched with a saturated aqueous NaHCO₃ solution. The crude product wasextracted with methylene chloride, washed with saturated aq NaHCO₃solution, and water. The combined organic phases were concentrated underreduced pressure, then dissolved in a mixture of dioxan (160 ml) andaqueous ammonia (28%, 29 ml). The solution was heated at 70° C. for 3 hand evaporated to dryness. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient of methanol [1-5%] inmethylene chloride) to give protected nucleoside 7 as a yellow solid(81%). R_(f)=0.16 (30% EtOAc in CH₂Cl₂) ¹H-NMR (400 MHz, DMSO-d₆) 1.03(s, 3H, CH₃), 1.30 (s, 3H, CH₃), 1.42 (s, 3H, CH₃), 3.5 (m, 2H, H-5′,H-5″), 3.71 (s, 6H, 2×OCH₃), 4.0 (d, 1H, H-4′, J=3.2 Hz), 4.36 (d, 1H,H-3′, J=2.8 Hz), 5.1 (m, 1H, OH-5′, D₂O exchangeable), 5.90 (s, 1H,H-1′), 6.2 (m, 1H, H-5), 6.8-7.2 (m, 13H, DMTr), 7.6 (m, 1H, H-6), 8.32(s, 1H, NH, D₂O exchangeable); LC/MS Scan ES− 598 (M−H)⁻, λ_(max1)=231.7nm, λ_(max2)=283.7 mm.

Synthesis of the Pronucleotide 10

Compounds 7 (2.0 g, 3.34 mmol) and 5 (2.2 eq, 4.3 g) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (50 ml).Pivaloyl chloride (2.5 eq, 1 ml) was added dropwise and the solutionstirred at room temperature for 2h30. The reaction mixture was dilutedwith methylene chloride and neutralized with an aqueous solution ofammonium chloride (NH₄Cl 0.5M). After extraction with methylenechloride/aq NH₄Cl 0.5M, the organic phases were combined, evaporatedunder reduced pressure (bath temperature not exceeding 30° C.) andcoevaporated with toluene. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride+2‰ acetic acid) to afford the desired product 8 whichwas coevaporated with toluene to give a beige foam (94%). R_(f)=0.63 (5%MeOH in CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) 1.21 (m, 9H, 3 CH₃), 1.42 (s,3H, CH₃), 1.60 (s, 3H, CH₃), 3.13 (m, 2H, CH₂S), 3.17 (m, 2H, CH₂OTr),3.79 (s, 6H, 2×OCH₃), 4.1 (m, 2H, CH₂OP), 4.2-4.3 (m, 3H, H-5′, H-5″,H-4′), 5.09 (d, 1H, H-3′, J=7.6 Hz), 5.89 (d, 1H, H-5, J=5.6 Hz), 6.0(m, 1H, H-1′), 6.8-7.7 (m, 29H, Tr, DMTr, H-6); ¹³P-NMR (161 MHz, CDCl₃)7.92, 8.55; LC/MS Scan ES+ 1066 (M+H)⁺, Scan ES− 1064 (M−H)⁻.

To a solution of compound 8 (3.4 g, 3.15 mmol) in anhydrous carbontetrachloride (30 ml) was added dropwise benzylamine (10 eq, 3.4 ml).The reaction mixture was stirred at room temperature for 1h30. A whiteprecipitate appeared. The solution was diluted with methylene chlorideand neutralized with an aqueous solution of hydrogen chloride (HCl 1M).After successive extractions with CH₂Cl₂/HCl 1M and CH₂Cl₂/aq NaHCO₃,the organic phases were combined, dried over Na₂SO₄, filtered andevaporated to dryness. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient [0-5%] of methanol inmethylene chloride) to give 2 as a yellow foam (87%). Rf=0.35 (5% MeOHin methylene chloride); ¹H-NMR (400 MHz, CDCl₃) 1.1-1.2 (m, 9H, 3 CH₃),1.40 (s, 3H, CH₃), 1.59 (s, 3H, CH₃), 2.9-3.2 (m, 4H, CH₂OTr, CH₂OS),3.76 (s, 6H, 2×OCH₃), 3.9-4.4 (m, 8H, CH₂OP, CH₂N, H-3′, H-4′, H-5′,H-5″), 5.0 (m, 1H, H-5), 6.0 (2s, 1H, H-1′), 6.7-7.7 (m, 34H, Tr, DMTr,C₆H₅CH₂, H-6); ¹³P-NMR (161 MHz, CDCl₃) 8.40, 8.8.68; LC/MS Scan ES+1171 (M+H)⁺.

Finally, compound 9 (2.39 g, 2.04 mmol) was dissolved in a mixture ofmethylene chloride (10 ml) and an aqueous solution of trifluoroaceticacid (90%, 10 ml). The reaction mixture was stirred at 35-40° C. for 2h, then diluted with ethanol (140 ml). The volatiles were evaporatedunder reduced pressure and coevaporated with ethanol. The crude mixturewas purified by silica gel column chromatography (eluant: stepwisegradient of methanol [0-30%] in methylene chloride), followed by apurification on reverse phase chromatography (eluant: stepwise gradientof acetonitrile [0-50%] in water), to give the desired product 10 (B102)(1:1 mixture of diastereoisomers as judged by ³¹P-NMR, 36%) which waslyophilized from a mixture of dioxan/water. Rf=0.34 (15% MeOH inmethylene chloride); ¹H-NMR (400 MHz, DMSO-d6) 0.92 (s, 3H, CH₃), 1.10(s, 6H, 2×CH₃), 3.0 (m, 2H, CH₂S), 3.33 (m, 1H, H-3′), 3.56 (s, 2H,CH₂OH), 3.8-4.0 and 4.05-4.25 (stacks, 7H, CH₂OP, NCH₂Ph, H-4′, H-5′ andH-5″), 4.9 (m, 1H, OH-3′, J=5.4 Hz, D₂O exchangeable), 5.07 (s, 1H,OH-2′, D₂O exchangeable), 5.3 (m, 1H, CH₂OH, D₂O exchangeable), 5.6-5.7(m, 2H, H-5 and NH, D₂O exchangeable), 5.91 (s, 1H, H-1′), 7.3-7.4(stack, 7H, PhH, NH₂, D₂O exchangeable), 7.6 (m, 1H, H-6); ¹³P-NMR (161MHz, DMSO-d6) 9.71, 9.91; HPLC t_(R)=4.67 min (0-100% acetonitrile overa period of 8 min), λ_(max)=274.9; LC/MS Scan ES+ 587 (M+H)⁺.

Strategy B

Synthesis of Protected Nucleoside 11

NM107 (10 g, 38.87 mmol) was dissolved in anhydrous pyridine (194 ml)and chlorotrimethylsilane (4.5 eq, 21.6 ml) was added. The reactionmixture was stirred at room temperature under nitrogen atmosphere for2h30, then 4,4′-dimethoxytrityl chloride (1.5 eq, 19.8 g) and4-dimethylaminopyridine (0.5 eq, 2.37 g) were successively added. Thereaction mixture was stirred overnight at room temperature, thenquenched with a saturated aqueous NaHCO₃ solution. The crude product wasextracted with methylene chloride, washed with saturated aq NaHCO₃solution, and water. The combined organic phases were concentrated underreduced pressure, then dissolved in tetrahydrofuran (110 ml). To thatsolution was added tetrabutylammonium fluoride 1M in THF (1 eq, 38.87ml) and the reaction mixture was stirred for 30 min at room temperature.After extraction with EtOAc and water, the organic phases were collectedand evaporated to dryness. The crude mixture was purified on silica gelcolumn chromatography (eluant: stepwise gradient of methanol [0-10%] inmethylene chloride) to give protected nucleoside 11 as a yellow solid(93%). R_(f)=0.32 (10% MeOH in CH₂Cl₂) ¹H-NMR (400 MHz, DMSO-d₆) 0.79(s, 3H, CH₃), 3.56 (m, 2H, H-5′, H-5″), 3.71 (s, 7H, 2×OCH₃, H-4′), 5.0(m, 4H, H-3′, OH-2′, OH-3′, OH-5′, D₂O exchangeable), 5.72 (s, 1H,H-1′), 6.16 (m, 1H, H-5), 6.8-7.2 (m, 13H, DMTr), 7.82 (m, 1H, H-6),8.24 (m, 1H, NH D₂O exchangeable); LC/MS Scan ES− 560 (M+H)⁺, ES− 558(M−H)⁻, λ_(max)=284.7 nm.

Synthesis of Protected Phosphoroamidate Pronucleotide 13, Precursor of10

Compound 11 (7 g, 12.5 mmol) and 5 (1.5 eq, 11.0 g) were coevaporatedtogether with anhydrous pyridine and dissolved in this solvent (187 ml).Pivaloyl chloride (2.0 eq, 3.08 ml) was added dropwise at −15° C. andthe solution stirred at this temperature for 1h30. The reaction mixturewas diluted with methylene chloride and neutralized with an aqueoussolution of ammonium chloride (NH₄Cl 0.5M). After extraction withmethylene chloride/aq NH₄Cl 0.5M, the organic phases were combined,evaporated under reduced pressure (bath temperature not exceeding 30°C.) and coevaporated with toluene. The crude mixture was purified onsilica gel column chromatography (eluant: stepwise gradient [0-5%] ofmethanol in methylene chloride+0.2% acetic acid) to afford the desiredproduct 12 which was coevaporated with toluene to give a white foam (3.5g, 27%). R_(f)=0.44 (5% MeOH in CH₂Cl₂); ¹H-NMR (400 MHz, DMSO) 0.8 (m,3H, CH₃), 1.14 and 1.06 (2s, 6H, 2 CH₃), 3.06 (m, 2H, CH₂S), 3.16 (m,2H, CH₂OTr), 3.5 (m, 1H, H-3′), 3.70 (m, 6H, 2 OCH₃), 3.90 (m, 1H,H-4′), 4.03 (m, 2H, CH₂OP), 4.24 (m, 2H, H-5′, H-5″), 5.30 and 5.04 (2ms, 2H, OH-2′ and OH-3′, D₂O exchangeable), 5.78 (m, 1H, H-1′), 5.98 (m,1H, P—H), 6.22 (m, 1H, H-5), 7.0-7.5 (m, 16H, Tr), 8.32 (m, 1H, H-6);¹³P-NMR (161 MHz, DMSO) 9.17, 9.65; LC/MS Scan ES+ 1026 (M+H)⁺,λ_(max)=282.7 nm.

To a solution of compound 12 (500 mg, 0.49 mmol) in anhydrous carbontetrachloride (4.9 ml) was added dropwise benzylamine (5 eq, 0.266 ml).The reaction mixture was stirred at room temperature for 3 h and thesolvent removed under reduced pressure. The crude mixture was purifiedon silica gel column chromatography (eluant: stepwise gradient [0-5%] ofmethanol in methylene chloride) to afford compound 13 as a foam (75%).Rf=0.25 (3% MeOH in methylene chloride); ¹H-NMR (400 MHz, DMSO) 0.79 (s,3H, CH₃), 1.13 and 1.06 (2s, 6H, 2 CH₃), 3.05 (m, 4H, CH₂OTr, CH₂OS),3.51 (m, 1H, H-3′), 3.69 (s, 6H, 2×OCH₃), 3.87 (m, 3H, CH₂OP, CH₂N,H-3′), 4.08 (m, 2H, H-5′, H-5″), 5.19 and 5.0 (2m, 2H, OH-2′ and OH-3′,D₂O exchangeable), 5.67 (m, 1H, NH, D₂O exchangeable), 5.75 (2s, 1H,H-1′), 6.21 (m, 1H, H-5), 6.7-7.5 (m, 34H, Tr, DMTr, C₆H₅CH₂, H-6);¹³P-NMR (161 MHz, DMSO) 9.84, 9.69; LC/MS Scan ES+ 1132 (M+H)⁺.

Compound 13 can be converted into the phosphoroamidate prodrug 10 (B102)following experimental conditions described for the last step of NM108-and NM105-OH-SATE phosphoroamidate synthesis, in Examples 3 and 4,respectively.

Example 3 Preparation of Hyrdoxy-t-BuSATE-N-BenzylphosphoramidateDerivative of 2′-C-Methylguanosine

Synthetic Strategy:

2′-C-methylguanidine (NM108) (3 g, 10.10 mmol) and compound 5 (6.48 g,11.10 mmol) were coevaporated together with anhydrous pyridine anddissolved in this solvent (152 mL). Pivaloyl chloride (2.48 mL, 20.18mmol) was added dropwise at −15° C. and the solution was stirred at thesame temperature for 2 h. The reaction mixture was diluted withmethylene chloride and neutralized with an aqueous solution of ammoniumchloride (NH₄Cl 0.5M). After extraction with methylene chloride/aq NH₄Cl0.5M, the organic phases were combined, dried over Na₂SO₄ evaporatedunder reduce pressure (bath temperature not exceeding 30° C.) andcoevaporated twice with toluene. The crude mixture was purified onsilica gel flash column chromatography (eluant: stepwise gradient[0-10%] of methanol in methylene chloride+0.2% acetic acid) to affordthe desired product 6 (2.5 g, 32%). R_(f)=0.34 (15% MeOH in CH₂Cl₂);¹H-NMR (400 MHz, DMSO-d₆ 0.80 (s, 3H, CH₃), 1.13 (s, 6H, 2×CH₃), 3.04(m, 2H, CH₂OTr), 3.14 (m, 2H, CH₂S), 3.97-4.08 (m, 4H, H-3′, H-4′,CH₂OP), 4.28-4.38 (m, 2H, H-5′, H-5″), 5.10-5.35 (m, 2H, OH-2′, OH-3′,D₂O exchangeable), 5.77 (s, 1H, H-1′), 6.52 (bs, 2H, NH₂, D₂Oexchangeable), 7.11-7.42 (m, 15H, Tr), 7.75 (s, 1H, H-8), 10.67 (bs, 1H,NH, D₂O exchangeable); ¹³P-NMR (161 MHz, DMSO-d₆) 9.47, 9.20; LC/MS ScanES+ 764 (M+H)⁺, Scan ES− 762 (M−H)⁻.

To a solution of compound 6 (2.5 g, 3.27 mmol) in anhydrous carbontetrachloride (33 mL) was added dropwise benzylamine (5 eq, 1.79 mL).The reaction mixture was stirred at room temperature for 1 h andevaporated under reduced pressure (bath temperature not exceeding 30°C.). The crude mixture was purified on silica gel flash columnchromatography (eluant: stepwise gradient [0-10%] of methanol inmethylene chloride) to give compound 7 as a white foam (2.9 g,quantitative yield). R_(f)=0.27 (10% MeOH in methylene chloride); ¹H-NMR(400 MHz, DMSO-d₆) 0.81 (s, 3H, CH₃), 1.10 (s, 6H, 2×CH₃), 2.99-3.08 (m,4H, CH₂OTr, CH₂S), 3.87-4.30 (m, 8H, H-3′, H-4′, H-5′, H-5″ CH₂OP,NCH₂Ph), 5.66 (m, 1H, NH, D₂O exchangeable), 5.76 (s, 1H, H-1′), 6.60(bs, 2H, NH₂, D₂O exchangeable), 7.17-7.39 (m, 20H, Tr, C₆H₅CH₂), 7.77(s, 1H, H-8); ¹³P-NMR (161 MHz, DMSO-d₆) 9.93, 9.78; LC/MS Scan ES+ 869(M+H)⁺, Scan ES− 867 (M−H)⁻.

Compound 7 (2.84 g, 3.27 mmol) was dissolved in a mixture oftrifluoroacetic acid (1.1 mL) and methylene chloride (11.4 mL). Thereaction mixture was stirred 0.5 h at room temperature. The solution wasdiluted with ethanol, evaporated under reduce pressure (bath temperaturenot exceeding 30° C.) and coevaporated twice with toluene. The crudemixture was purified on silica gel flash column chromatography (eluant:stepwise gradient [0-30%] of methanol in methylene chloride) and then,on reverse phase column chromatography (eluant: stepwise gradient[0-100%] of acetonitrile in water) to give the desired product 8 (B184)(1:1 mixture of diastereoisomers according to ³¹P-NMR, 800 mg, 39%)which was lyophilized from a mixture of dioxan/water. Rf=0.57 (20% MeOHin methylene chloride); ¹H-NMR (400 MHz, DMSO-d6) 0.82 (s, 3H, CH₃),1.09 (s, 6H, 2×CH₃), 3.01 (m, 2H, CH₂S), 3.42 (d, 2H, CH₂OH, J=8.0 Hz),3.81-4.00 (m, 6H, H-3′, H-4′ CH₂OP, NCH₂Ph), 4.11-4.27 (m, 2H, H-5′,H-5″), 4.92 (t, 1H, CH₂OH, J=8.0 Hz, D₂O exchangeable), 5.16 (s, 1H,OH-2′, D₂O exchangeable), 5.40 (m, 1H, OH-3′, D₂O exchangeable), 5.64(m, 1H, NH, D₂O exchangeable), 5.75 (s, 1H, H-1′), 6.50 (bs, 2H, NH₂ D₂Oexchangeable), 7.19-7.32 (m, 5H, PhH), 7.77 (s, 1H, H-8), 10.61 (bs, 1H,NH, D₂O exchangeable); 13P-NMR (161 MHz, DMSO-d6) 9.91, 9.78; HPLCt_(R)=3.67 min (0-100% acetonitrile over a period of 8 min),λ_(max)=251.3; LC/MS Scan ES+ 627 (M+H)⁺, Scan ES− 625 (M−H)⁻.

Example 4

A anti-cancer drug, R—OH, such as an antiviral nucleoside, having a freeOH group, is derivatized to form a phosphoramidate compound according tothe following scheme. Reactive groups on the molecule, such as otherhydroxyl groups, are protected using methods known in the art.

Example 5

A phosphoroamidate of 5-azacytidine is prepared as follows:

Examples 6-10 illustrate by way of example the effect of thephosphoroamidate group on an antiviral compound to promote liverspecific delivery of an active agent to liver cells.

Example 6 Preparation of Calibration Curve

Measurements of the concentration of2′-3′-dideoxyadenosine-5′-triphosphate (ddATP) (the triphosphatenucleotide of 2′-3′-dideoxyadenosine (ddA) are performed by liquidchromatography tandem mass spectrometry (LC/MS/MS), e.g., of methanolicextracts of hepatocytes.

The concentration of ddATP is measured by comparison to a standardcurve. Working stock solutions of TP-ddA are prepared from a 100 μmol/μlstock solution in de-ionized water of ddATP (tetrasodium salt of >91%purity) purchased from Sigma Chemical Co as follows:

ddATP Working Stock Solutions and Preparation of Standard Curve forddATP.

Stock Vol DIH₂O Total conc taken vol vol Conc mol per pmol/μl μL μL μLpmol/μl 10 μl 1. Working stock#1 Test compound TP-ddA 100 2000 2000 400050.0 500 2. Working stock#2 Test article TP-ddA 100 1000 3000 4000 25.0250 3. Working stock#4 (prepared from stock#1) TP-ddA 100 500 3500 400012.5 125 4. Working stock#5 (prepared from stock#1) TP-ddA 100 200 38004000 5.0 50 5. Working stock#6 (prepared from stock#1) TP-ddA 100 1003900 4000 2.5 25 6. Working stock#7 (prepared from stock#1) TP-ddA 10040 3960 4000 1.0 10

Internal standard (ISTD) working stock are prepared from a 0.50 mg/mLstock solution of 2-deoxyadenosine 5-triphosphate purchased from SigmaChemical Co.

Stock conc Vol taken MeOH vol Total vol Conc Conc ISTD μg/mL μL μL μLμg/mL pmol/mL dATP 500 200 9800 10000 10 500

In some embodiments, calibration standards are prepared as follows usingliver

Preparation of cal stds: std conc working working stock con workingstock vol ISTD vol MeOH vol total vol cal std# pmol/ml liver wt G stock#pmol/μL uL uL uL uL Blk 0 0.1 0 50 940 990 #1 50 0.1 #5 5.0 10 50 9401000 #2 125 0.1 #4 12.5 10 50 940 1000 #3 250 0.1 #3 25.0 10 50 940 1000#4 500 0.1 #2 50.0 10 50 940 1000 #5 1000 0.1 #1 100.0 10 50 940 1000samples:

In some embodiments the following HPLC conditions are used for the HPLCMS, e.g. HPLC Tandem MS analysis instrument method:

HPLC is conducted on Phenomenex Luna Amino 3 μm 100 A, 30×2 mm column,with a mobile phase: A: 70% 10 mM NH₄OAc 30% ACN pH 6.0; and B: 70% 1 mMNH₄OAc 30% ACN pH 10.5 as follows:

Time Flow Step (min) (μl/min) A (%) B (%) Gradient elution program: 0  0400 60  40 1  1.1 400 60  40 2  1.11 400 40  60 3  2.11 400 30  70 4 2.6 400 20  80 5  3.1 400  0 100 6  5.5 400  0 100 7  5.51 400 60  40 810 400 60  40 Injection volume: 50 ul Flow rate to MS: 0.400 mL/min, nosplitting of flow Multiple Reaction Monitoring (MRM) conditions:(API3000) Ionization Mode: Positive Ion Electrospray (ESI+) IonSprayVoltage (IS): 5000 V Temperature (TEM): 550° C. Turbo IS gas 8 L/minNebulizer (NEB): 14 CAD Gas Setting (CAD):  6 Declustering potential(DP) 68 V Collision energy (CE) 27 eV Entrance/Exit potentials 10 V/11 V(EP/CXP) Compound Precursor ion => Product Ion ddA triphosphate 476.2 =>135.9 ddA diphosphate 396.2 => 135.9 dA triphosphate (ISTD) 460.2 =>135.9*Luna Amino column is directly connected on the inlet end to a “SecurityGuard” cartridge holder suitable for 2.1 mm Phenomenex columns,containing a C18 cartridge.

Example 7 In Vitro Phosphorylation in Hepatocytes

Primary hepatocytes (Rat, Cynomolgus Monkey or human) were seeded at0.8×10⁶ in a collagen-coated 12-well plate and allowed to attach 4-6hours after which time the seeding medium was replaced with serum-freeculture medium and cells allowed to acclimatize to the new mediumovernight. On the next day, cells were exposed for 1, 4, 8 and 24 hoursto test article A550 (NM204) at 10 and 50 μM prepared in fresh culturemedium from stock solution in DMSO (final DMSO concentration was 0.1%).At each time point, an aliquot (500 μl) was collected and immediatelyadded to 500 μl of acetonitrile and stored at −20° C. until analysis.The remaining exposure medium was removed and the cell monolayer (stuckto dish) washed 2 times with ice-cold PBS. Any remaining PBS wascarefully removed by aspiration and cells were harvested by scraping in1 mL 70% ice-cold methanol. Cell samples were placed overnight at −20°C. and cellular debris removed by centrifugation on the next day. Thesupernatants were removed and filtered prior to analysis by LC/MS. Astandard curve was prepared by using untreated cells processed similarlyexcept that prior to harvesting in 70% methanol, 10 μl of LddATPstandard solutions prepared in methanol were added to the washedmonolayers. These control samples were then processed and analyzed asdescribed for test samples.

The results are shown below:

LddA-TP formation in hepatocytes LddA TP Levels (pmol/million cells) RatMonkey Human A550 (Ex 1) 10 μM Time (hour) 1 159.5 287.5 161.5 4 388.0978.0 312.5 8 468.5 1230.0 352.5 24  422.0 344.0 366.0 A550 (Ex 1) 50 μMTime (hour) 1 393.0 2085.0 682.5 4 1212.0 5690.0 1480.0 8 1590.0 6030.01930.0 24  1505.0 3030.0 2062.5

As indicated from the data, significant levels of L-ddATP were detectedin the hepatocytes. In monkeys, the levels appear to reach a maximumlevel at 8 hours followed by a rapid decline. In contrast, levels inboth rat and human hepatocyte appear to level off after 8 hours.

Example 8 In Vivo Studies in Rat

Distribution of A550 (NM-204) (the compound of Example 1(Hydroxy-tBuSATE N-benzylphosphoroamidate derivative of L-ddA) in therat liver was evaluated following a single intravenous (I.V.) or oraladministration of A550 (NM-204) at a dose of 20 (oral) or 10 (I.V.)mg/Kg body weight. The dose solutions were prepared on the same dayprior to dose administration.

At the specified time point (1 and 3 hours for IV animals or 1, 3 and 8hours for oral animals), each animal was euthanized by CO₂ gas followedby exsanguination via the abdominal vein. Livers were collectedimmediately after sacrifice, flash frozen in liquid nitrogen, placed ondry ice, and later stored at −70° C., before being analyzed.

Preparation of Calibration Standards from Control Liver Extracts:

Control rat liver samples were taken from whole frozen livers(Bioreclamation, Inc. Hicksville, N.Y.) with the aid of a tissue coringutensil (Harris Unicore, 8.0 mm, VWR). Each ˜0.1 g sample was placed inindividual 2 mL poly vials with 0.940 mL of 80% MeOH/20% DIH₂O andhomogenates were prepared using a mechanical tissue disruptor (TissueMaster, Omni-International, Inc, Marietta Ga.). The vials received a 10μl aliquot of a working stock solution and a 50 μl aliquot of the ISTDbefore vortexing for ˜30 sec. The mixtures were stored overnight at −20°C. and the next day were removed for 10 min of centrifugation in abenchtop centrifuge. Each supernatant was transferred to individualcentrifugation filtration units (0.45 μm) and the resulting filtrateswere transferred to HPLC vials for the LC/MS/MS analysis. The finalconcentrations of ddATP in the calibration standards was 1000, 500, 250,125, 50, and 0 pmol/ml. Each calibration standard was directly injectedin a 50 μL volume onto the ion-exchange column for analysis. Standardcurve analysis of calibration standards from control liver extracts wasconducted.

Analysis of ddATP was done by an ion-exchange chromatography method withon-line positive ionization ESI-MS/MS detection in multiple reactionmonitoring (MRM) detection mode. The peak areas obtained for 4 of the 5calibrants allowed for construction of a standard curve thatdemonstrated good linearity (R²=0.9996) over a 50-1000 μmol/mlconcentration range. This is equivalent to a range of 5-100 μmol pergram liver by the sample preparation employed. The HPLC MS MS conditionsdescribed in Example 5 were utilized. The lower limit of quantitationdemonstrated by the LC/MS/MS method is e.g., ˜0.2 pmol/mL for hepatocytecellular extracts which contain much less salt.

The results showing intracellular levels of A550 (NM204) (showing thecompound entered the liver cells) and LddATP (showing cleaving of thephosphoroamidate moiety and triphosphorylation of the ddA to the activetriphosphate in the liver) are shown below:

A550 (Ex 1) and LddATP measured in livers of male rats dosed IV or Owith A550 (Ex 1) Concentration Concentration. Compound ddA-TP (Ex 1)Timepoint (pmol/ (pmol/10⁶ Animal Number (pmol/g liver) (hrs) g liver)cells)* IV dose (10 mg/kg) 1M1 65.8 1 2025 17.8 1M2 89.1 1 1930 16.9 1M385.1 1 1355 11.9 Mean 80.0 1770 15.5 IV dose (10 mg/kg) 2M1 28.3 3 134511.8 2M2 26.0 3 1940 17.0 2M3 29.3 3 2990 26.2 Mean 27.9 2092 18.3 Oraldose (20 mg/kg) 3M1 411 1 210 1.8 3M2 272 1 575 5.0 3M3 70.2 1 400 3.5Mean 251 395 3.5 Oral dose (20 mg/kg) 4M1 360 3 200 1.8 4M2 92.1 3 3302.9 4M3 161 3 405 3.6 Mean 204 312 2.7 Oral dose (20 mg/kg) 5M1 16.4 8280 2.5 5M2 28 8 805 5.2 5M3 16.2 8 275 2.4 Mean 20.1 382 3.3*Hepatocellularity number for rat was 114 × 106 cells per gram liver(Toxicology in Vitro 20 (2005) 1582-1586.

Thus, these results show that the compound can be used to enhanceconcentration of the drug in the liver. These results also show theenhanced concentration of the active triphosphate which is formed in theliver cells.

Example 9 Determination of Total Metabolism in Liver SubcellularFractions Depletion of Parent

NADPH Incubations. Microsomal or S9 incubations were conducted in afinal volume of 0.5 mL. Pooled liver microsomal or S9 protein (1.0mg/mL), suspended in incubation buffer (100 mM potassium phosphate, pH7.4, 5 mM MgCl₂, and 0.1 mM EDTA) was preincubated for 5 min at 37° C.with 10-50 μM OHSATE phosphoroamidate compound from a stock solution inDMSO (final DMSO concentration was 0.1%); the reaction was initiated bythe addition of NADPH (3 mM final concentration). Incubations with noNADPH served as controls. At specific times (0-120 min), 0.1 mL sampleswere taken and the reaction terminated by the addition of 1 volume ofstop solution (acetonitrile). The samples were vortex for 30 sec andthen centrifuged at 1500 g for 10 min. The supernatant was transferredto HPLC glass vials and analyzed without further processing by HPLC.FIGS. 1 and 2 depict depletion of NM108 SATE and NM107 SATE,respectively, after incubation with NADPH in monkey liver S9.

HPLC System for Medium Samples-Unchanged Prodrug

HPLC: Agilent 1100 Column: Phenomenex Luna C18(2), 20 × 2 mm, Mobilephases (MP): MP(A) 10 mM K₂HPO₄ pH5, MP(B) ACN Gradient elution: 20 to63% MP(B) run from 0 to 30 min Runtime: 20 min Flow rate: 1 mL/minInjection volume: 10-20 μL UV: 252 nm-NM108SATE 272 nm-NM107SATE

Thus, without being limited to any theory, since the metabolism is NADPHdependent, it is possible that the phosphoroamidate compound ispreferentially activated by Cytochrome P450 in the liver.

Example 10 Determination of Triphosphate Levels in Cells

Preparation of Primary Hepatocyte Cultures

Freshly isolated cells from animal and human liver were obtained insuspension on ice. Following receipt, cells were pelleted bycentrifugation at 500 rpm (rat) or 700 rpm (monkey and human) andresuspended at 0.8 million cells per mL of platting medium (HPM).Multi-well collagen-coated plates (12-well) were then seeded by additionof 1 mL of cell suspention (0.8 million cells/mL). The plates weregently shaken to evenly distribute the cells and placed in an incubatorat 37° C. for approximately 4 to 6 hours to allow cells to attach. Oncecells have attached, the platting medium was removed and replaced withhepatocyte culture medium (HCM). Cells were left overnight in anincubator at 37° C. to acclimatize to culture and the medium.

Incubations with Test Article

Hepatocyte incubations were conducted in a final volume of 1.0 mLHCM/well (0.8 million cells/mL). HCM from overnight incubation of cellswas removed and replaced with fresh HCM, pre-warmed to 37° C.,containing 10 μM test article from a stock solution in DMSO (final DMSOconcentration was 0.1%). At specific times (up to 24 hrs), incubationmedium was discarded and the cell monolayers were carefully washed twotimes with ice-cold PBS. Following the last wash, all PBS was carefullyremoved and 1 mL of extraction buffer (ice-cold 70% methanol) was added.Each well was sealed with parafilm immediately following addition ofmethanol. Once the entire plate was processed, additional parafilm wasplaced on entire plate forming a double seal to prevent evaporationduring the extraction process. The cover lid was then placed on theplate and sealed with tape. The plates were then stored at −20° C. for aminimum of 24 hrs to allow for extraction of intracellular contents.

Preparation of Huh7 or HepG2 Cultures

HepG2s or Huh7 cells were plated at 0.4×10⁶ cells/well incollagen-coated 12-well plates. Cells were allowed to attach overnight.Culture medium from overnight incubation of cells was removed andreplaced with fresh culture medium, pre-warmed to 37° C., containing 10μM test article from a stock solution in DMSO (final DMSO concentrationwas 0.1%). After 24-72 hours, incubation medium was discarded and thecell monolayers were carefully washed two times with ice-cold PBS.Following the last wash, all PBS was carefully removed and 1 mL ofextraction buffer (ice-cold 70% methanol) was added. Each well wassealed with parafilm immediately following addition of methanol. Oncethe entire plate was processed, additional parafilm was placed on entireplate forming a double seal to prevent evaporation during the extractionprocess. The cover lid was then placed on the plate and sealed withtape. The plates were then stored at −20° C. for a minimum of 24 hrs toallow for extraction of intracellular contents.

Sample Preparation for Analysis

Cellular extracts were prepared by transferring 0.9 mL of extract into 2mL microfuge tubes followed by centrifugation for 5 min at 14,000 rpm.Approximately 100 μL of the supernatant was transferred to HPLC vialsand triphosphate levels determined by LCMS/MS as described below.

HPLC conditions: NM107-triphosphate

HPLC: Column: Phenomenex Luna Amino 3 μm 100A, 30 × 2 mm, Mobile phases(MP): (A) 70% 10 mM NH₄OAc 30% ACN pH 6.0 (B) 70% 1 mM NH₄OAc 30% ACN pH10.5 Step Time Flow A B Gradient elution: 0 0.00 400 80  20 1 0.10 40080  20 2 0.11 400 40  60 3 0.21 400 40  60 4 2.60 400 10  90 5 2.61 400 0 100 6 5.60 400  0 100 7 5.61 400 80  20 8 9.00 400 80  20 Flow rateto MS: 0.400 mL/min, no split Injection volume: 10 μL Compound Precursorion Product ion NM107 triphosphate 498.0 112.0

HPLC conditions: NM108-triphosphate

HPLC: Column: Phenomenex Luna Amino 3 pm 100A, 30 × 2 mm, Mobile phases(MP): (A) 70% 10 mM NH₄OAc 30% ACN pH 6.0 (B) 70% 1 mM NH₄OAc 30% ACN pH10.5 Step Time Flow A B Gradient elution: 0 0.00 400 60  40 1 0.10 40060  40 2 0.11 400 40  60 3 0.21 400 40  60 4 2.60 400 10  90 5 2.61 400 0 100 6 5.61 400  0 100 7 5.61 400 60  40 8 9.00 400 60  40 Flow rateto MS: 0.400 mL/min, no split Injection volume: 10 μL Compound Precursorion Product ion NM108 triphosphate 538.0 152.0

NM107 triphosphate levels and B102 in cell extracts were observed asfollows:

Intracellular Triphosphate (pmol/million cells) drug in culture HumanMonkey HepG2* Huh7* B102 (Ex. 2) 991 1838 1.5 9.2 NM107 19 10 17 37 24hr incubation in 10 μM drug *72 hr incubation in 10 μM drug

As seen from the data levels of intracellular triphosphate for B102 (Ex.2) were much higher as compared to those for NM107.

All publications and patent, applications cited in this specificationare herein incorporated by reference as if each individual publicationor patent application were specifically and individually indicated to beincorporated by reference. While the claimed subject matter has beendescribed in terms of various embodiments, the skilled artisan will

1. A compound of formula

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof, wherein: R^(y) is optionallysubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,cycloalkenyl, amino, heterocyclyl or heteroaryl; R^(a) and R^(b) areselected as follows: i) R^(a) and R^(b) are each independently hydrogenor optionally substituted alkyl, carboxyalkyl, hydroxyalkyl,hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl, alkoxycarbonylalkyl,aryl, arylalkyl, cycloalkyl, heteroaryl or heterocyclyl; or ii) R^(a)and R^(b) together with the nitrogen atom on which they are substitutedform a 3-7 membered heterocyclic or heteroaryl ring; and R¹ is a moietyderivable by removal of a hydrogen from a hydroxy group of ananti-cancer drug.
 2. The compound of claim 1, having the formula:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof.
 3. The compound of claim 1having the formula:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof.
 4. The compound of claim 1having the formula:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof.
 5. The compound of claim 1,wherein R^(y) is optionally substituted alkyl and R^(a) and R^(b) areeach independently hydrogen or optionally substituted benzyl.
 6. Thecompound of claim 5, wherein R^(y) is hydroxyalkyl or aminoalkyl.
 7. Thecompound of claim 1, wherein R^(y) is —C(R^(c))₃ or —NHR^(c) where eachR^(c) is independently optionally substituted alkyl or optionallysubstituted aryl; and R^(a) and R^(b) are independently hydrogen,optionally substituted alkyl or optionally substituted arylalkyl.
 8. Thecompound of claim 7, wherein R^(a) and R^(b) are each independentlyhydrogen or substituted alkyl.
 9. The compound of claim 5, wherein R^(y)is selected from the group consisting of alkyl and hydroxyalkyl.
 10. Thecompound of claim 9, wherein R^(y) is —C(CH₃)₂CH₂OH.
 11. The compound ofclaim 9, wherein R^(a) is hydrogen, R^(b) is benzyl and R^(y) is—C(CH₃)₂CH₂OH.
 12. The compound of any of claims 1, wherein R¹ isAclarubicin, Decitabine, Daunorubicin, Dihydro-5-azacytidine,Doxorubicin, Epirubicin, Estramustine, Etoposide, Fludarabine,Neplanocin A, Tezacitabine, Troxacitabine, Vinblastin, Vincristin,Vindesin, Teniposide, NK-611, Camptothecin, Irinotecan,9-Aminocamptothecin, Topotecan, Paclitaxel, Azatoxin, Coformycin,Pirarubicin, or Losoxantrone.
 13. A compound of formula:

wherein each R, if present, is independently alkyl, halogen or hydroxyl;X, if present, is CH₂, O or S; R^(y) is alkyl, alkenyl, alkynyl, aryl,aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl, heterocyclyl orheteroaryl, all optionally substituted; R^(a) and R^(b) are selected asfollows: i) R^(a) and R^(b) are each independently hydrogen, alkyl,carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl,heteroaryl or heterocyclyl, all optionally substituted; or ii) R^(a) andR^(b) together with the nitrogen atom on which they are substituted forma 3-7 membered heterocyclic or heteroaryl ring.
 14. The compound ofclaim 13, wherein each R, if present, is independently alkyl, halogen orhydroxyl; X, if present, is CH₂, O or S; R^(y) is optionally substitutedalkyl, wherein the substituents when present are hydroxy or amino; andR^(a) and R^(b) are each independently hydrogen or alkyl, wherein thealkyl group is optionally substituted with aryl, amino, amido, hydroxyl,alkoxy or heteroaryl, each optionally substituted.
 15. The compound ofclaim 13, wherein R^(a) and R^(b) are each independently H or benzyl,wherein the benzyl group is optionally substituted with hydroxy oramino.
 16. The compound of claim 13, wherein R^(y) is substituted alkyland R^(a) and R^(b) are each independently hydrogen or optionallysubstituted benzyl.
 17. The compound of claim 16, wherein R^(y) ishydroxyalkyl or aminoalkyl.
 18. The compound of claim 13, wherein R^(y)is —C(R^(c))₃ or —NHR^(c) where each R^(c) is independently optionallysubstituted alkyl or optionally substituted aryl; and R^(a) and R^(b)are independently hydrogen, optionally substituted alkyl or optionallysubstituted arylalkyl.
 19. The compound of claim 17, wherein R^(y) is—C(CH₃)₂CH₂OH.
 20. The compound of claim 19, wherein R^(a) and R^(b) areeach independently hydrogen or substituted alkyl.
 21. The compound ofclaim 13, wherein R^(a) is hydrogen, R^(b) is benzyl and R^(y) is—C(CH₃)₂CH₂OH.
 22. The compound of claim 13 having the structure:

or a pharmaceutically acceptable salt, solvate, a stereoisomeric,tautomeric or polymorphic form thereof.
 23. A compound selected fromformula:

wherein R^(x) and R^(z) are each independently hydrogen or optionallysubstituted alkyl; R^(w) is optionally substituted alkyl; X¹ is O or S;R^(y) is alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,cycloalkenyl, amino, heterocyclyl or heteroaryl, all optionallysubstituted; R^(a) and R^(b) are selected as follows: i) R^(a) and R^(b)are each independently hydrogen, alkyl, carboxyalkyl, hydroxyalkyl,hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl, alkoxycarbonylalkyl,aryl, arylalkyl, cycloalkyl, heteroaryl or heterocyclyl, all optionallysubstituted; or ii) R^(a) and R^(b) together with the nitrogen atom onwhich they are substituted form a 3-7 membered heterocyclic orheteroaryl ring.
 24. The compound of claim 23, wherein R^(y) isoptionally substituted alkyl, wherein the substituents when present areselected from hydroxy and amino.
 25. The compound of claim 24, whereinR^(y) is —C(CH₃)₂CH₂OH.
 26. The compound of claim 25, wherein R^(a) ishydrogen and R^(b) is benzyl.
 27. The compound of claim 26 havingformula selected from:


28. The compound of claim 27 having formula:


29. A method of treating cancer comprising administering a compound ofclaim
 1. 30. A method of lowering plasma lipid levels or lowering bloodglucose levels comprising administering a compound of claim
 13. 31. Amethod of lowering blood glucose levels comprising administering acompound of claim
 23. 32. A pharmaceutical composition comprising acompound of any of claims 1, 13 or 23 and a pharmaceutically acceptablecarrier.
 33. The composition of claim 32 that is suitable for oraladministration.
 34. The composition of claim 33 wherein the compositionis in the form of a pill or tablet.